FfAME:org :: All FfAME Publications

All FfAME Publications

Functional Selection of Tau Oligomerization-Inhibiting Aptamers
Bang Wang, Xiaoshu Pan, I-Ting Teng, Xiaowei Li, Firas Kobeissy, Zo-Yu Wu, Jiepei Zhu, Guangzheng Cai, He Yan, Xin Yan, Mingwei Liang, Fahong Yu, Zunyi Yang, Elisa Biondi, William Haskins, Y. Charles Cao, Steven A. Benner, Weihong Tan, Kevin Wang
Angew. Chem. Int. Ed. (2024) e202402007, https://doi.org/10.1002/anie.202402007
<Abstract>

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd = 6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3β-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Enzyme-assisted high throughput sequencing of an expanded genetic alphabet at single base resolution
Bang Wang, Kevin M. Bradley, Myong-Jung Kim, Roberto Laos, Cen Chen, Dietlind L. Gerloff, Luran Manfio, Zunyi Yang & Steven A. Benner
Nat. Commun.15(4057), Nature (2024) https://doi.org/10.1038/s41467-024-48408-9
<Abstract>

With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created "artificially expanded genetic information systems" (AEGIS) to add nucleotides, functionality, and information density. With the expected improvements seen in AegisBodies and AegisZymes, the task for synthetic biologists shifts to developing for expanded DNA the same analytical tools available to natural DNA. Here we report one of these, an enzyme-assisted sequencing of expanded genetic alphabet (ESEGA) method to sequence six-letter AEGIS DNA. We show how ESEGA analyses this DNA at single base resolution, and applies it to optimized conditions for six-nucleotide PCR, assessing the fidelity of various DNA polymerases, and extending this to AEGIS components with functional groups. This supports the renewed exploitation of expanded DNA alphabets in biotechnology.

A folding motif formed with an expanded genetic alphabet
Bang Wang, James R. Rocca, Shuichi Hoshika, Cen Chen, Zunyi Yang, Reza Esmaeeli, Jianguo Wang, Xiaoshu Pan, Jianrong Lu, Kevin K. Wang, Y. Charles Cao, Weihong Tan & Steven A. Benner
Nat. Chem., Nature (2024) https://doi.org/10.1038/s41557-024-01552-7
<Abstract>

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5°C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ^-:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ^-:dZ pairs join parallel strands in a four-stranded compact down-up-down-up fold. These have two possible structures: one with intercalated dZ^-:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC⁺-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

The chemistry of formycin biosynthesis
Richards NGJ, Naismith JN
Front. Chem. Biol., Frontiers Media SA (2024) 11 July: 3, 1428646. DOI: 10.3389/fchbi.2024.1428646
<Abstract>

Remarkable progress has been made to elucidate the structural and mechanistic enzymology of the biosynthetic pathways that give rise to naturally occurring C-nucleosides. These compounds are generally cytotoxic and exhibit interesting antiviral, antibiotic and anti-parasitic activity. Here we review current knowledge concerning formycin biosynthesis and highlight deficiencies in our understanding of key chemical transformations in the pathway.

Arginine kinase activates arginine for phosphorylation by pyramidalization and polarization
Falcioni F, Molt RW Jr., Jin Y, Waltho JP, Hay S, Richards, NGJ, Blackburn GM
ACS Catal, ACS (2024) 16 April: 14(9), 6650-6658. DOI: 10.1021/acscatal.4c00380
<Abstract>

Arginine phosphorylation plays numerous roles throughout biology. Arginine kinase (AK) catalyzes the delivery of an anionic phosphoryl group (PO₃^–) from ATP to a planar, trigonal nitrogen in a guanidinium cation. Density functional theory (DFT) calculations have yielded a model of the transition state (TS) for the AK-catalyzed reaction. They reveal a network of over 50 hydrogen bonds that delivers unprecedented pyramidalization and out-of-plane polarization of the arginine guanidinium nitrogen (Nη2) and aligns the electron density on Nη2 with the scissile P–O bond, leading to in-line phosphoryl transfer via an associative mechanism. In the reverse reaction, the hydrogen-bonding network enforces the conformational distortion of a bound phosphoarginine substrate to increase the basicity of Nη2. This enables Nη2 protonation, which triggers PO₃^– migration to generate ATP. This polarization–pyramidalization of nitrogen in the arginine side chain is likely a general phenomenon that is exploited by many classes of enzymes mediating the post-translational modification of arginine.

Reactivity and mechanism in chemical and synthetic biology
Richards, NGJ, Bearne SL, Goto Y, Parker EJ
Phil. Trans. R. Soc. B Biol. Sci., The Royal Society Publishing (2023) 27 February: 378(1871), 20220023. DOI: 10.1098/rstb.2022.0023
<Abstract>

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria. The COVID-19 pandemic led to postponement of in-person discussions, with the symposium eventually being held on 20–21 December 2021 as an online event. This issue is a written record of work presented on biosynthetic pathways and enzyme catalysis, engineering microorganisms with new metabolic capabilities, and the synthesis of non-canonical, nucleobases for medical applications and for studies of alternate chemistries for living organisms. The variety of opinion pieces, reviews and original research articles provide a starting point for innovations that clarify how complex biological systems emerge from the rules of chemical reactivity and mechanism.

Experimental and computational snapshots of C-C bond formation in a C-nucleoside synthase
Li W, Girt GC, Radadiya A, Stewart JJP, Richards NGJ, Naismith JN
Open Biology, The Royal Society Publishing (2023) 11 January: 13(1), 220287. DOI: 10.1098/rsob.220287
<Abstract>

The biosynthetic enzyme, ForT, catalyses the formation of a C-C bond between 4-amino-1H-pyrazoledicarboxylic acid and MgPRPP to produce a C-nucleoside precursor of formycin A. The transformation catalysed by ForT is of chemical interest because it is one of only a few examples in which C-C bond formation takes place via an electrophilic substitution of a small, aromatic heterocycle. In addition, ForT is capable of discriminating between the aminopyrazoledicarboxylic acid and an analogue in which the amine is replaced by a hydroxyl group; a remarkable feat given the steric and electronic similarities of the two molecules. Here we report biophysical measurements, structural biology and quantum chemical calculations that provide a detailed molecular picture of ForT-catalysed C-C bond formation and the conformational changes that are coupled to catalysis. Our findings set the scene for employing engineered ForT variants in the biocatalytic production of novel, anti-viral C-nucleoside and C-nucleotide analogues.

Fluorinated oil-surfactant mixtures with the density of water: Artificial cells for synthetic biology
Roberto Laos, Steven Benner
PLOS One17(1), Public Library of Science (2022) https://doi.org/10.1371/journal.pone.0252361
<Abstract>

There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span® 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman® assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".

Mist and replication
Rajamani, S., Biondi, E.
Nature Physics, Springer (2022) https://doi.org/10.1038/s41567-022-01549-4
<Abstract>

The transition from chemistry to evolvable molecular systems is at the core of origins of life studies. Now, the acidic dew–liquid water dynamic cycling inside simulated Hadean rock pores is found to possibly provide a confined environment for strand separation, replication, mutation, and the evolution of nucleic acids.

Catalytic Synthesis of Polyribonucleic Acid on Prebiotic Rock Glasses
Craig A. Jerome, Hyo-Joong Kim, Stephen J. Mojzsis, Steven A. Benner, and Elisa Biondi
Astrobiology (2022) http://doi.org/10.1089/ast.2022.0027
<Abstract>

Reported here are experiments that show that ribonucleoside triphosphates are converted to polyribonucleic acid when incubated with rock glasses similar to those likely present 4.3-4.4 billion years ago on the Hadean Earth surface, where they were formed by impacts and volcanism. This polyribonucleic acid averages 100-300 nucleotides in length, with a substantial fraction of 3',-5'-dinucleotide linkages. Chemical analyses, including classical methods that were used to prove the structure of natural RNA, establish a polyribonucleic acid structure for these products. The polyribonucleic acid accumulated and was stable for months, with a synthesis rate of 2 x 10^-3 pmoles of triphosphate polymerized each hour per gram of glass (25°C, pH 7.5). These results suggest that polyribonucleotides were available to Hadean environments if triphosphates were. As many proposals are emerging describing how triphosphates might have been made on the Hadean Earth, the process observed here offers an important missing step in models for the prebiotic synthesis of RNA.

2'-β-Selenium Atom on Thymidine to Control β-Form DNA Conformation and Large Crystal Formation
Cen Chen, Ziyuan Fang, and Zhen Huang
Cryst. Growth Des, ACS (2022) 22, 6, 3601-3604, DOI:10.1021/acs.cgd.2c00474
<Abstract>

Crystallization phasing and obtaining high-quality crystals are bottleneck challenges for the X-ray crystallographic analysis of nucleic acids, especially when dynamic behavior is to be inferred from crystallographic B-factors. Crystallization of DNA duplexes, normally existing in the B-form in solution, is especially challenging, as the high salt used in many crystallization processes favors their transformation to A-form DNA duplexes. To address crystallization challenges while avoiding structural perturbation, we explored atom-specific incorporation to place a selenium atom on the 2'-β (arabino) position of the 2'-deoxyribose ring. This incorporation is expected to favor the B-form of a DNA duplex during crystallization. Here, we report the first synthesis of the β-2'-MeSe-thymidine (^SeT) nucleoside, the corresponding Se-phosphoramidite, and Se-containing DNA oligonucleotides. We found that particular Se-DNAs form crystals that are surprisingly larger than we have often observed, having higher quality and giving improved diffraction resolution when compared to crystals from analogous standard oligonucleotides. Surprisingly, one duplex made from a self-complementary Se-containing oligonucleotide gave crystals 600 × 200 µm² in size; this is 10–100 times larger in volume than the corresponding crystals grown from native DNA. CD and a solved crystal structure showed that the selenium in the β-orientation did not perturb the native structure and gave diffraction data from which dynamic information could be extracted. Crystals of this size are especially important for neutron diffraction studies. Moreover, we discovered that the high-quality Se-DNA crystals diffracted to 1.15 Å. The Se-derivatized structure was virtually identical with the native structure. These discoveries suggest a simple strategy to address other crystallization challenges in nucleic acid crystallography, a strategy whose scope deserves further exploration.

Agnostic Life Finder (ALF) for Large-Scale Screening of Martian Life During In Situ Refueling
Spacek, J. & Benner, S.A.
Astrobiology (2022) 22, 8, DOI:10.1089/ast.2021.0070
<Abstract>

Before the first humans depart for Mars in the next decade, hundreds of tons of martian water-ice must be harvested to produce propellant for the return vehicle, a process known as in situ resource utilization (ISRU). We describe here an instrument, the Agnostic Life Finder (ALF), that is an inexpensive life-detection add-on to ISRU. ALF exploits a well-supported view that informational genetic biopolymers in life in water must have two structural features: (1) Informational biopolymers must carry a repeating charge; they must be polyelectrolytes. (2) Their building blocks must fit into an aperiodic crystal structure; the building blocks must be size-shape regular. ALF exploits the first structural feature to extract polyelectrolytes from ?10 cubic meters of mined martian water by applying a voltage gradient perpendicularly to the water's flow. This gradient diverts polyelectrolytes from the flow toward their respective electrodes (polyanions to the anode, polycations to the cathode), where they are captured in cartridges before they encounter the electrodes. There, they can later be released to analyze their building blocks, for example, by mass spectrometry or nanopore. Upstream, martian cells holding martian informational polyelectrolytes are disrupted by ultrasound. To manage the (unknown) conductivity of the water due to the presence of salts, the mined water is preconditioned by electrodialysis using porous membranes. ALF uses only resources and technology that must already be available for ISRU. Thus, life detection is easily and inexpensively integrated into SpaceX or NASA ISRU missions.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern
Nguyen, L. T., Macaluso, N. C., Pizzano, B. L., Cash, M. N., Spacek, J., Karasek, Jain, P. K., et. al.
EBioMedicine, Lancet (2022) 77, 103926. DOI:10.1016/j.ebiom.2022.103926

In vitro evolution of ribonucleases from expanded genetic alphabets
Jerome, C.A; Hoshika, S.; Bradley, K.M.; Benner, S.A.; Biondi, E.
Proc. Natl. Acad. Sci. USA (2022) 119(44). DOI: 10.1073/pnas.2208261119
<Abstract>

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucleotide building blocks is limited when compared with that of proteins. Here, we assess the evolutionary potential of libraries of nucleic acids with six nucleotide building blocks as reservoirs for catalysis. We compare the outcomes of in vitro selection experiments toward RNA-cleavage activity of two nucleic acid libraries: one built from the standard four independently replicable nucleotides and the other from six, with the two added nucleotides coming from an artificially expanded genetic information system (AEGIS). Results from comparative experiments suggest that DNA libraries with increased chemical diversity, higher information density, and larger searchable sequence spaces are one order of magnitude richer reservoirs of molecules that catalyze the cleavage of a phosphodiester bond in RNA than DNA libraries built from a standard four-nucleotide alphabet. Evolved AEGISzymes with nitro-carrying nucleobase Z appear to exploit a general acid–base catalytic mechanism to cleave that bond, analogous to the mechanism of the ribonuclease A family of protein enzymes and heavily modified DNAzymes. The AEGISzyme described here represents a new type of catalysts evolved from libraries built from expanded genetic alphabets.

Development of triazole-conjugated dihydropyrimidinone (DHPM) derivatives as potential P-glycoprotein inhibitors
Bijani S, Shaikh F, Mirza D, Siu SWI, Jain N, Ferreira RJ, dos Santos DJVA, Rawal R, Richards NGJ, Shah A, Radadiya A
ACS Omega, ACS (2022) 17 May: 7(19), 16278-16287. DOI: 10.1021/acsomega.1c05839
<Abstract>

P-glycoprotein (Pgp), an ATP binding cassette (ABC) transporter, is an ATP-dependent efflux pump responsible for cancer multidrug resistance. As part of efforts to identify human Pgp (hPgp) inhibitors, we prepared a series of novel triazole-conjugated dihydropyrimidinones using a synthetic approach that is well suited for obtaining compound libraries. Several of these dihydropyrimidinone derivatives modulate human P-glycoprotein (hPgp) activity with low micromolar EC50 values. Molecular docking studies suggest that these compounds bind to the M-site of the transporter.

Thermodynamic Parameters and Design Considerations for Expanded Alphabet PZ and GZ Base Pairs in DNA
Kahn, J. D., Miffin, T., Sharp, K. K., Wang, X., Hoshika, S., Sun, H., Benner, S. A., Mathews, D. H.
Biophys. J., AP Cell Press (2021) 120(3):220a, DOI:10.1016/j.bpj.2020.11.1475
<Abstract>

As expanded nucleic acid alphabets such as the AEGIS system become more prevalent in molecular biology, synthetic biology and DNA design, it becomes more essential to be able to make reliable predictions for the thermodynamics of secondary structures that include these new base pairs. Using previous work as well as new UV absorbance melting curves for DNA oligonucleotide duplexes, we have generated new nearest-neighbor parameters for the free energy, enthalpy, and entropy of all dinucleotide steps that include P:Z base pairs. The results are similar but show clear differences from previous work. We have also determined the nearest neighbor values for all dinucleotide steps containing the stable G:Z mismatch, as well as parameters for dangling ends and terminal mismatches containing P and Z nucleotides. Finally, we show that including P and Z bases in DNA design algorithms makes target structures easier to design and more robust (in silico), and that it is essential to include consideration of the G:Z pair to avoid stable unwanted structures and thereby achieve the improved performance.

The role of thrifty genes in the origin of alcoholism: A narrative review and hypothesis
Carn, D., Lanaspa, M. A., Benner, S. A., Andrews, P., Dudley, R., Tolan, D. R., Johnson, R. J.
Alcohol.: Clin. Exp. Res., Wiley-Blackwell (2021) 10.1111/acer.14655
<Abstract>

In this narrative review, we present the hypothesis that key mutations in two genes, occurring 15 and 10 million years ago (MYA), were individually and then collectively adaptive for ancestral humans during periods of starvation, but are maladaptive in modern civilization (i.e., "thrifty genes"), with the consequence that these genes not only increase our risk today for obesity, but also for alcoholism. Both mutations occurred when ancestral apes were experiencing loss of fruit availability during periods of profound climate change or environmental upheaval. The silencing of uricase (urate oxidase) activity 15 MYA enhanced survival by increasing the ability for fructose present in dwindling fruit to be stored as fat, a consequence of enhanced uric acid production during fructose metabolism that stimulated lipogenesis and blocked fatty acid oxidation. Likewise, a mutation in class IV alcohol dehydrogenase ~10 MYA resulted in a remarkable 40-fold increase in the capacity to oxidize ethanol (EtOH), which allowed our ancestors to ingest fallen, fermenting fruit. In turn, the EtOH ingested could activate aldose reductase that stimulates the conversion of glucose to fructose, while uric acid produced during EtOH metabolism could further enhance fructose production and metabolism. By aiding survival, these mutations would have allowed our ancestors to generate more fat, primarily from fructose, to survive changing habitats due to the Middle Miocene disruption and also during the late-Miocene aridification of East Africa. Unfortunately, the enhanced ability to metabolize and utilize EtOH may now be acting to increase our risk for alcoholism, which may be yet another consequence of once-adaptive thrifty genes.

Ultra-rapid detection of SARS-CoV-2 in public workspace environments
Yaren, O., McCarter, J., Phadke, N., Bradley, K. M., Overton, B., Yang, Z., Ranade, S., Patil, K., Bangale, R., Benner, S. A.
PLOS One, Public Library of Science (2021) 10.1371/journal.pone.0240524, DOI:10.1101/2020.09.29.20204131
<Abstract>

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 10⁵ virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.

Abiotic Synthesis of Nucleoside 5'-Triphosphates with Nickel Borate and Cyclic Trimetaphosphate (CTMP)
Kim, H.J.., Benner, S.A.
Astrobiology (2021) 21(3), DOI:10.1089/ast.2020.2264
<Abstract>

While nucleoside 5'-triphosphates are precursors for RNA in modern biology, the presumed difficulty of making these triphosphates on Hadean Earth has caused many prebiotic researchers to consider other activated species for the prebiotic synthesis of RNA. We report here that nickel(II), in the presence of borate, gives substantial amounts (2–3%) of nucleoside 5'-triphosphates upon evaporative heating in the presence of urea, salts, and cyclic trimetaphosphate (CTMP). Also recovered are nucleoside 5'-diphosphates and nucleoside 5'-monophosphates, both likely arising from 5'-triphosphate intermediates. The total level of 5'-phosphorylation is typically 30%. Borate enhances the regiospecificity of phosphorylation, with increased amounts of other phosphorylated species seen in its absence. Experimentally supported paths are already available to make nucleosides in environments likely to have been present on Hadean Earth soon after a midsized 1021 to 1023 kg impactor, which would also have delivered nickel to the Hadean surface. Further, sources of prebiotic CTMP continue to be proposed. Thus, these results fill in one of the few remaining steps needed to demystify the prebiotic synthesis of RNA and support a continuous model from atmospheric components to oligomeric RNA that is lacking only a mechanism to obtain homochirality in the product RNA.

Molecular Dynamics and Machine Learning in Catalysts
Wenxiang Liu, Yang Zhu, Yongqiang Wu, Cen Chen, Yang Hong, Yanan Yue, Jingchao Zhang, Bo Hou
Catalysts, MDPI (2021) 11:1129, DOI:10.3390/catal11091129
<Abstract>

Given the importance of catalysts in the chemical industry, they have been extensively investigated by experimental and numerical methods. With the development of computational algorithms and computer hardware, large-scale simulations have enabled influential studies with more atomic details reflecting microscopic mechanisms. This review provides a comprehensive summary of recent developments in molecular dynamics, including ab initio molecular dynamics and reaction force-field molecular dynamics. Recent research on both approaches to catalyst calculations is reviewed, including growth, dehydrogenation, hydrogenation, oxidation reactions, bias, and recombination of carbon materials that can guide catalyst calculations. Machine learning has attracted increasing interest in recent years, and its combination with the field of catalysts has inspired promising development approaches. Its applications in machine learning potential, catalyst design, performance prediction, structure optimization, and classification have been summarized in detail. This review hopes to shed light and perspective on ML approaches in catalysts.

A simple electroanalysis of polyG RNA in mixtures with 3', 5'-cyclic guanosine monophosphate achieved by selective desorption of the monomers from the electrode surface
Hesko, O., Fojta, M., & Spacek, J.
J. Electroanal. Chem, Elsevier (2021) 901, 115773. DOI: 10.1016/j.jelechem.2021.115773
<Abstract>

Previously it has been shown that cyclic nucleoside monophosphates can spontaneously polymerize to form RNA oligonucleotides under conditions simulating prebiotic conditions on Archean Earth. The most efficient polymerization was documented with 3',5'-cyclic guanosine monophosphate (cGMP). In this work a method for fast detection of short polyG RNAs present in a large overabundance of cGMP, modeling conditions in the non-enzymatic nucleotide polymerization mixtures, is presented. The method is based on electrochemical measurements of guanine (G) oxidation signals yielded by RNA oligomers adsorbed onto the surface of a pyrolytic graphite electrode (PGE). To avoid false positive results arising from the G oxidation signals due to co-adsorbed cGMP, a method for selective removal of the monomers from the electrode surface has been devised. In the first step, both cGMP and RNAs are co-adsorbed onto the PGE surface. In the second step, the cGMP is selectively desorbed using treatments in solutions of different tested surfactants (SDS, Tween 20 or Triton X-100), or by washing in deionized water at elevated temperature. We show that this new approach is suitable for selective analysis of products of polymerization reactions from mixtures of their building blocks.

The Limits to Organic Life in the Solar System: From Cold Titan to Hot Venus
Benner, S. A., & Spacek, J.
LPI Contributions, LPI (2021) 2629, 4003.
<Abstract>

Synthetic biology, physical organic chemistry, and other experimental sciences help assess here on Earth the probability of life in alien environments, and guide our search for it in observational and mission astronomy. These have given us "Agnostic Life Finders" (ALFs) for worlds where environments are similar to Earth's. However, they also guide a search for life in exotic environments. We will present by lab experiments showing organic processes that may support life in acidic Venusian clouds.

Functional characterization of two PLP-dependent enzymes involved in capsular polysaccharide biosynthesis from Campylobacter jejuni
Riegert AS, Narindoshvili T, Coricello A, Richards NGJ, Raushel FM
Biochemistry, ACS (2021) 21 September: 60(19), 2836-2843. DOI: 10.1021/acs.biochem.1c00439
<Abstract>

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Building better polymerases: Engineering the replication of expanded genetic alphabets
Ouaray, Z., Benner, S. A., Georgiadis, M. M., Richards, N. G. J.
J. Biol. Chem., ASBMB (2020) 11 December: 295(50):17046-17059, DOI:10.1074/jbc.REV120.013745
<Abstract>

DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Improving the treatment of acute lymphoblastic leukemia
Radadiya A, Zhu W, Coricello A, Alcaro S, Richards NGJ
Biochemistry, ACS (2020) 8 September: 59(35), 3193-3200. DOI: 10.1021/acs.biochem.0c00354
<Abstract>

l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.

Uncovering the chemistry of C-C bond formation in C-nucleoside biosynthesis: Crystal structure of a C-glycoside synthase/PRPP complex
Gao S, Radadiya A, Li W, Liu H, Zhu W, de Crecy-Lagard V, Richards NGJ, Naismith JN
Chem. Comm., Royal Society of Chemistry (2020) 14 July: 56(55), 7617-7620. DOI: 10.1039/d0cc02834g
<Abstract>

The enzyme ForT catalyzes C–C bond formation between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5'-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.

Tautomeric equilibria of nucleobases in the hachimoji expanded genetic alphabet
Eberlein, L., Beierlein, F.R., van Eikema Hommes, N.J.R., Radadiya, A., Heil, J., Benner, S.A., Clark, T., Kast, S.M., and Richards, N.G.J.
J. Chem. Theory Comput., ACS (2020) 14 Apr: 16(4):2766-2777, DOI:10.1021/acs.jctc.9b01079
<Abstract>

Evolution has yielded biopolymers that are constructed from exactly four building blocks and are able to support Darwinian evolution. Synthetic biology aims to extend this alphabet, and we recently showed that 8-letter (hachimoji) DNA can support rule-based information encoding. One source of replicative error in non-natural DNA-like systems, however, is the occurrence of alternative tautomeric forms, which pair differently. Unfortunately, little is known about how structural modifications impact free-energy differences between tautomers of the non-natural nucleobases used in the hachimoji expanded genetic alphabet. Determining experimental tautomer ratios is technically difficult, and so, strategies for improving hachimoji DNA replication efficiency will benefit from accurate computational predictions of equilibrium tautomeric ratios. We now report that high-level quantum-chemical calculations in aqueous solution by the embedded cluster reference interaction site model, benchmarked against free-energy molecular simulations for solvation thermodynamics, provide useful quantitative information on the tautomer ratios of both Watson-Crick and hachimoji nucleobases. In agreement with previous computational studies, all four Watson-Crick nucleobases adopt essentially only one tautomer in water. This is not the case, however, for non-natural nucleobases and their analogues. For example, although the enols of isoguanine and a series of related purines are not populated in water, these heterocycles possess N1-H and N3-H keto tautomers that are similar in energy, thereby adversely impacting accurate nucleobase pairing. These robust computational strategies offer a firm basis for improving experimental measurements of tautomeric ratios, which are currently limited to studying molecules that exist only as two tautomers in solution.

Whole-genome sequence of Streptomyces kaniharaensis Shomura and Niida SF-557
Zhu W, Liu X, Hughes M, de Crecy-Lagard V, Richards NGJ
Microbiol. Resource Announ., ASM (2020) Apr: 9(14), e01434. DOI: 10.1128/MRA.01434-19
<Abstract>

Streptomyces kaniharaensis is a Gram-positive bacterium that produces formycin A 5'-phosphate, a C nucleotide with antimicrobial and anticancer activity. Here, we report the sequencing, assembly, and annotation of the draft genome sequence of Streptomyces kaniharaensis Shomura and Niida.

Building better enzymes: Molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase
Ouaray Z, Singh I, Georgiadis MM, Richards NGJ
Protein Sci., The Protein Society (2020) Feb: 29(2), 455-468. DOI: 10.1002/pro.3762
<Abstract>

Obtaining semisynthetic microorganisms that exploit the information density of "hachimoji" DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the "hachimoji" P:Z nucleobase pair with a similar efficiency to that seen for Watson-Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the "hachimoji" Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.

When Did Life Likely Emerge on Earth in an RNA-First Process?
S. A. Benner, E. A. Bell, E. Biondi, R. Brasser, T. Carell, H.-J. Kim, S. J. Mojzsis, A. Omran, M. A. Pasek, D. Trail
ChemSystemsChem2, Chemistry Europe (2020) e1900035
<Abstract>

The widespread presence of ribonucleic acid (RNA) catalysts and cofactors in the Earth's biosphere today suggests that RNA was the first biopolymer to support Darwinian evolution. However, most "path-hypotheses" to generate building blocks for RNA require reduced nitrogen-containing compounds not made in useful amounts in the CO2-N2-H2O atmospheres of the Hadean. We review models for Earth's impact history that invoke a single ~1023 kg impactor (Moneta) to account for measured amounts of platinum, gold, and other siderophilic ("iron-loving") elements on the Earth and Moon. If it were the last sterilizing impactor, by reducing the atmosphere but not the mantle Moneta, would have opened a "window of opportunity" for RNA synthesis, a period when RNA precursors rained from the atmosphere onto land holding oxidized minerals that stabilize advanced RNA precursors and RNA. Surprisingly, this combination of physics, geology, and chemistry suggests a time when RNA formation was most probable, ~120±100 million years after Moneta's impact, or ~4.36±0.1 billion years ago. Uncertainties in this time are driven by uncertainties in rates of productive atmosphere loss and amounts of sub-aerial land.

An Aptamer-Nanotrain Assembled from Six-Letter DNA Delivers Doxorubicin Selectively to Liver Cancer Cells.
Zhang, L., Wang, S., Yang, Z., Hoshika, S., Xie, S., Li, J., Chen, X., Wan, S., Li, L., Benner, S.A., Tan, W.
Angew. Chem. Int. Ed. (2020) 59(2): 663-668, DOI:10.1002/anie.201909691
<Abstract>

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six-letter GACTZP DNA contributes this property in two parts of a nanoassembly: (1) in an aptamer evolved from a six-letter DNA library to selectively bind liver cancer cells; and (2) in a six-letter self-assembling GACTZP nanotrain that carries the drug doxorubicin. The aptamer-nanotrain assembly, charged with doxorubicin, selectively kills liver cancer cells in culture, as the selectivity of the aptamer binding directs doxorubicin into the aptamer-targeted cells. The assembly does not kill untransformed cells that the aptamer does not bind. This architecture, built with an expanded genetic alphabet, is reminiscent of antibodies conjugated to drugs, which presumably act by this mechanism as well, but with the antibody replaced by an aptamer.

Confluence of Theory and Experiment Reveal the Catalytic Mechanism of the Varkud Satellite Ribozyme
Ganguly, A., Weissman, B.P., Giese, T.J., Li, N.-S. , Hoshika, S., Rao, S., Benner, S.A., Piccirilli, J.A., York, D.M.
Nat. Chem., Nature (2020) 12:193-201, DOI:10.1038/s41557-019-0391-x
<Abstract>

The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab initio quantum mechanical/molecular mechanical free-energy simulations to gain insight into the catalysis. Through this confluence of theory and experiment, we unify the existing body of structural and functional data to unveil the catalytic mechanism in unprecedented detail, including the degree of proton transfer in the transition state. Further, we provide evidence for a critical Mg²⁺ in the active site that interacts with the scissile phosphate and anchors the general base guanine in position for nucleophile activation. This novel role for Mg²⁺ adds to the diversity of known catalytic RNA strategies and unifies functional features observed in the Varkud satellite, hairpin and hammerhead ribozyme classes.

Ligand Guided Selection (LIGS) with Artificially Expanded Genetic Information Systems against TCR-CD3ε
Zumrut, H., Yang, Z., Williams, N., Arizala, J., Batool, S., Benner, S.A., Mallikaratchy, P.
Biochemistry, ACS (2020) 59(4):552-562, DOI:10.1021/acs.biochem.9b00919
<Abstract>

Here we are reporting, for the first time, a ligand-guided selection (LIGS) experiment using an artificially expanded genetic information system (AEGIS) to successfully identify an AEGIS–DNA aptamer against T cell receptor-CD3ε expressed on Jurkat.E6 cells. Thus, we have effectively combined the enhanced diversity of an AEGIS DNA library with LIGS to develop a superior screening platform to discover superior aptamers. Libraries of DNA molecules from highly diversified building blocks will provide better ligands due to more functional diversity and better-controlled folding. Thus, a DNA library with AEGIS components (dZ and dP) was used in LIGS experiments against TCR-CD3ε in its native state using two clinically relevant monoclonal antibodies to identify an aptamer termed JZPO-10, with nanomolar affinity. Multiple specificity assays using knockout cells, and competition experiments using monoclonal antibodies utilized in LIGS, show unprecedented specificity of JZPO-10, suggesting that the combination of LIGS with AEGIS-DNA libraries will provide a superior screening platform to discover artificial ligands against critical cellular targets.

Science Addressing Questions Not Easily Addressed by "the" Scientific Method. The Origins of Life
Benner, S.A.
PAS Acta, The Pontifical Academy of Sciences (2020) 25:123-161

Eliminating Primer Dimers and Improving SNP detection using Self-Avoiding Molecular Recognition Systems (SAMRS)
Yang, Z., Le, J.T., Hutter, D., Bradley, K.M., Overton, B.R., McLendon, C., Benner, S.A.
Biol. Methods Protoc., Oxford Academics (2020) 5(1):bpaa004, DOI:10.1093/biomethods/bpaa004
<Abstract>

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer-primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

Chemical guidance in the search for past and extant life on Mars. Decadal Survey in Planetary Sciences and Astrobiology
Benner, S. A., Biondi, E., Kim, H.-J., Spacek, J.
Bulletin of the AAS, AAS (2020) 53(4), DOI:10.3847/25c2cfeb.266df7e7
<Abstract>

NASA should design missions to Mars to generate "Aha!" jolts for scientists researching the molecular origins of life. Recent advances allow these missions to be informed via privileged chemistry that likely generated RNA prebiotically on Earth, as well as general rules that constrain the structure of genetic molecules of extant life on Mars.

Electrochemical Reduction and Oxidation of Eight Unnatural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode
Spacek, J., Karalkar, N., Fojta, M., Wang, J., Benner, S. A
Electrochimica acta, International Society of Electrochemistry (2020) 362:137210, DOI:10.1016/j.electacta.2020.137210
<Abstract>

Recently we showed the reduction and oxidation of six natural 2'-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2'-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

Electroanalysis of unnatural base pair content in plasmid DNA generated in a semi-synthetic organism
Spacek, J., & Fojta, M.
Electrochimica acta, International Society of Electrochemistry (2020) 364, 137298. DOI: 10.1016/j.electacta.2020.137298
<Abstract>

While standard Watson-Crick base pairing normally involves hydrogen bonds that join size complementary nitrogenous heterocycles, certain unnatural base pairs (UBP) can support replication, transcription, and translation, including these in semi-synthetic organisms (SSO), using size complementarity alone. In this paper we show that the UBPs can be analyzed electrochemically as components of ribo- or 2'-deoxyribonucleosides, corresponding (2'-deoxy)triphosphates, as well as incorporated into DNA. Here, performance of the electrochemical methods profits from the ability of the unnatural bases 5SICS and TPT3 adsorbed at the surface of a mercury electrode to catalyze hydrogen evolution in acidic media. This allows semi-quantitative detection of single UBP within plasmid DNA generated in a SSO. This electrochemical approach provides new way for direct, fast, and low cost UBP analysis and can supplement or replace currently used methods of UBP detection.

Tautomeric equilibria of nucleobases in the hachimoji expanded genetic alphabet
Eberlein L, Beierlein F, van Eikema-Hommes, NJR, Radadiya A, Heil J, Benner SA, Clark, T, Kaast SM, Richards NGJ
J. Chem. Theory Comput., ACS (2020) 14 Apr: 16(4), 2766-2777. DOI: 10.1021/acs.jctc.9b01079
<Abstract>

Evolution has yielded biopolymers that are constructed from exactly four building blocks and are able to support Darwinian evolution. Synthetic biology aims to extend this alphabet, and we recently showed that 8-letter (hachimoji) DNA can support rule-based information encoding. One source of replicative error in non-natural DNA-like systems, however, is the occurrence of alternative tautomeric forms, which pair differently. Unfortunately, little is known about how structural modifications impact free-energy differences between tautomers of the non-natural nucleobases used in the hachimoji expanded genetic alphabet. Determining experimental tautomer ratios is technically difficult, and so, strategies for improving hachimoji DNA replication efficiency will benefit from accurate computational predictions of equilibrium tautomeric ratios. We now report that high-level quantum-chemical calculations in aqueous solution by the embedded cluster reference interaction site model, benchmarked against free-energy molecular simulations for solvation thermodynamics, provide useful quantitative information on the tautomer ratios of both Watson–Crick and hachimoji nucleobases. In agreement with previous computational studies, all four Watson–Crick nucleobases adopt essentially only one tautomer in water. This is not the case, however, for non-natural nucleobases and their analogues. For example, although the enols of isoguanine and a series of related purines are not populated in water, these heterocycles possess N1–H and N3–H keto tautomers that are similar in energy, thereby adversely impacting accurate nucleobase pairing. These robust computational strategies offer a firm basis for improving experimental measurements of tautomeric ratios, which are currently limited to studying molecules that exist only as two tautomers in solution.

Hachimoji DNA and RNA: A genetic system with eight building blocks
Hoshika H, Leal N, Kim MJ, Kim MS, Karalkar NB, Kim HJ, Bates AM, Watkins Jr. NE, SantaLucia HA, Meyer AJ, DasGupta S, Piccirilli JA, Ellington AD, SantaLucia Jr. J, Georgiadis MM, Benner SA
Science (2019) 22 Feb 2019: Vol. 363, Issue 6429, pp. 884-887. DOI: 10.1126/science.aat0971
<Abstract>

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

The surprising pairing of 2-aminoimidazo[1,2-a]- [1,3,5]triazin-4-one, a component of an expanded DNA alphabet
Roberto Laos, Christos Lampropoulos, and Steven A. Benner
Structural Chemistry, Acta Crystallographica (2019) C75, 22-28, https://doi.org/10.1107/S2053229618016923
<Abstract>

Synthetic biologists demonstrate their command over natural biology by reproducing the behaviors of natural living systems on synthetic biomolecular platforms. For nucleic acids, this is being done stepwise, first by adding replicable nucleotides to DNA, and then removing its standard nucleotides. This challenge has been met in vitro with 'six-letter' DNA and RNA, where the Watson-Crick pairing 'concept' is recruited to increase the number of independently replicable nucleotides from four to six. The two nucleobases most successfully added so far are Z and P, which present a donor-donor-acceptor and an acceptor-acceptor- donor pattern, respectively. This pair of nucleobases are part of an 'artificially expanded genetic information system' (AEGIS). The Z nucleobase has been already crystallized, characterized, and published in this journal [Matsuura et al. (2016). Acta Cryst. C72, 952-959]. More recently, variants of Taq polymerase have been crystallized with the pair P:Z trapped in the active site. Here we report the crystal structure of the nucleobase 2-aminoimidazo[1,2-a][1,3,5]- triazin-4-one (trivially named P) as the monohydrate, C5H5N5O-H2O. The nucleobase P was crystallized from water and characterized by X-ray diffraction. Interestingly, the crystal structure shows two tautomers of P packed in a Watson-Crick fashion that cocrystallized in a 1:1 ratio.

Prebiotic Chemistry that Could Not Not Have Happened
Benner S.A., Kim H.-J., and Biondi E.
Life9(4), MDPI 84 (2019) https://doi.org/10.3390/life9040084
<Abstract>

We present a direct route by which RNA might have emerged in the Hadean from a fayalite-magnetite mantle, volcanic SO2 gas, and well-accepted processes that must have created substantial amounts of HCHO and catalytic amounts of glycolaldehyde in the Hadean atmosphere. In chemistry that could not not have happened, these would have generated stable bisulfite addition products that must have rained to the surface, where they unavoidably would have slowly released reactive species that generated higher carbohydrates. The formation of higher carbohydrates is self-limited by bisulfite formation, while borate minerals may have controlled aldol reactions that occurred on any semi-arid surface to capture that precipitation. All of these processes have well-studied laboratory correlates. Further, any semi-arid land with phosphate should have had phosphate anhydrides that, with NH3, gave carbohydrate derivatives that directly react with nucleobases to form the canonical nucleosides. These are phosphorylated by magnesium borophosphate minerals (e.g., luneburgite) and/or trimetaphosphate-borate with Ni2+ catalysis to give nucleoside 5'-diphosphates, which oligomerize to RNA via a variety of mechanisms. The reduced precursors that are required to form the nucleobases came, in this path-hypothesis, from one or more mid-sized (1023-1020 kg) impactors that almost certainly arrived after the Moon-forming event. Their iron metal content almost certainly generated ammonia, nucleobase precursors, and other reduced species in the Hadean atmosphere after it transiently placed the atmosphere out of redox equilibrium with the mantle. In addition to the inevitability of steps in this path-hypothesis on a Hadean Earth if it had semi-arid land, these processes may also have occurred on Mars. Adapted from a lecture by the Corresponding Author at the All-Russia Science Festival at the Lomonosov Moscow State University on 12 October 2019, and is an outcome of a three year project supported by the John Templeton Foundation and the NASA Astrobiology program. Dedicated to David Deamer, on the occasion of his 80th Birthday.

Hydroxymethanesulfonate from volcanic sulfur dioxide. A mineral reservoir for formaldehyde in prebiotic chemistry.
Kawai, J., McLendon, D.C., Kim, H.-J., Benner, S.A.
Astrobiology (2019) 19(4):506-516, DOI:10.1089/ast.2017.1800
<Abstract>

While formaldehyde (HCHO) was likely generated in Earth's prebiotic atmosphere by ultraviolet light, electrical discharge, and/or volcano-created lightning, HCHO could not have accumulated in substantial amounts in prebiotic environments, including those needed for prebiotic processes that generate nucleosidic carbohydrates. HCHO at high concentrations in alkaline solutions self-reacts in the Cannizzaro reaction to give methanol and formate, neither having prebiotic value. Here, we explore the possibility that volcanic sulfur dioxide (SO₂) might have generated a reservoir for Hadean HCHO by a reversible reaction with HCHO to give hydroxymethanesulfonate (HMS). We show that salts of HMS are stable as solids at 90°C and do not react with themselves in solution, even at high (>8 M) concentrations. This makes them effective stores of HCHO, since the reverse reaction slowly delivers HCHO back into an environment where it can participate in prebiotically useful reactions. Specifically, we show that in alkaline borate solutions, HCHO derived from HMS allows formation of borate-stabilized carbohydrates as effectively as free HCHO, without losing material to Cannizzaro products. Further, we show that SO2 can perform similar roles for glycolaldehyde and glyceraldehyde, two intrinsically unstable carbohydrates that are needed by various models as precursors for RNA building blocks. Zircons from the Hadean show that the Hadean mantle likely provided volcanic SO2 at rates at least as great as the rates of atmospheric HCHO generation, making the formation of Hadean HMS essentially unavoidable. Thus, hydroxymethylsulfonate adducts of formaldehyde, glycolaldehyde, and glyceraldehyde, including the less soluble barium, strontium, and calcium salts, are likely candidates for prebiotically useful organic minerals on early Earth.

A Prebiotic Synthesis of Canonical Pyrimidine and Purine Ribonucleotides
Kim, H.-J., Kim, J.
Astrobiology (2019) 19(5):669-74, DOI:10.1089/ast.2018.1935
<Abstract>

The "RNA first" model for the origin of life holds that RNA emerged spontaneously on early Earth and developed into life through its dual capabilities for genetics and catalysis. The model's central weakness is the difficulty of making its building blocks, in particular, the glycosidic bond joining nucleobases to ribose. Thus, the focus of much of the modern literature on the topic is directed toward solving this difficulty and includes elegant, though indirect, methods for making this bond. Here, we report that the glycosidic bond in canonical pyrimidine and purine ribonucleotides can be formed by direct coupling of cyclic carbohydrate phosphates with free nucleobases, all reported to be available by experimentally supported pathways that might have operated on early Earth.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and and four serotypes of dengue viruses in mosquitoes.
Glushakova, L.G.. Alto, B.W., Kim, M.-S., Hutter, D., Bradley, A., Bradley, K.M., Burkett-Cadena, N.D., Benner, S.A.
BMC Infect. Dis., BioMed Central Ltd. (2019) 19:418, DOI:10.1186/s12879-019-3998-z
<Abstract>

Background The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.

Methods We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).

Results The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.

Conclusions Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

Electrochemical Reduction and Oxidation of Six Natural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode in the Presence or Absence of Ambient Oxygen
Spacek, J., Fojta, M., & Wang, J.
Electroanalysis, Wiley (2019) 31(10), 2057-2066. DOI: 10.1002/elan.201900417
<Abstract>

Recently we have demonstrated that pyrolytic graphite electrodes (PGE) offer a broad analytically useful potential window, ranging from about -2.0V to +1.6V vs. Ag|AgCl|3M KCl, which enables to use the PGE not only for anodic measurements, but also for direct reduction of nucleobases in DNA oligonucleotides. In this follow-up study, we have focused on the electrochemical behavior of four 2'-deoxynucleosides derived from adenine, guanine, cytosine, thymine, uracil, and 5-methyl cytosine on the PGE. On one hand we have obtained analogous primary redox responses as previously with the oligonucleotides. On the other hand, significant differences were observed, particularly when considering secondary responses involving products of the primary conversions, suggesting involvement of different mechanisms. Further we have found that presence of the ambient oxygen in the electrolyte does not dramatically affect the redox signals of the nucleosides. This finding is in contrast with DNA responses measured at the mercury-based electrodes, where deaeration prior to the measurements was necessary. We demonstrate that all studied nucleosides can be analyzed using a simple ex situ (medium exchange) procedure.

Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography
Deepak Koirala, Sandip A. Shelke, Marcel Dupont, Stormy Ruiz, Saurja DasGupta, Lucas J. Bailey, Steven A. Benner and Joseph A. Piccirilli
Nucl. Acids Res. 1-12 (2018) doi: 10.1093/nar/gkx1292
<Abstract>

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ~100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.

A Direct Prebiotic Synthesis of Nicotinamide Nucleotide
Hyo-Joong Kim, Steven A. Benner
Chemistry, Wiley-VCH (2018) Jan 12;24(3):581-584. doi: 10.1002/chem.201705394
<Abstract>

Under the "RNA World" hypothesis, an early episode of natural history on Earth used RNA as the only genetically encoded molecule to catalyze steps in its metabolism catalysis. This, according to the hypothesis, included RNA catalysts that used RNA cofactors. However, the RNA World hypothesis places special demands on prebiotic chemistry, which must now deliver not only four ribonucleosides, but also must deliver the "functional" portion of these RNA cofactors. While some (e.g. methionine) present no particular challenges, nicotinamide ribose is special. Essential to its role in biological oxidations and reductions, its glycosidic bond that holds a positively charged heterocycle is especially unstable with respect to cleavage. Nevertheless, we are able to report here a prebiotic synthesis of phosphorylated nicotinamide ribose under conditions that also conveniently lead to the adenosine phosphate components of this and other RNA cofactors.

Artificially Expanded Genetic Information Systems for New Aptamer Technologies
Elisa Biondi and Steven A. Benner
Biomedicines, MDPI (2018) 6, 53; doi:10.3390/biomedicines6020053
<Abstract>

Directed evolution was first applied to diverse libraries of DNA and RNA molecules a quarter century ago in the hope of gaining technology that would allow the creation of receptors, ligands, and catalysts on demand. Despite isolated successes, the outputs of this technology have been somewhat disappointing, perhaps because the four building blocks of standard DNA and RNA have too little functionality to have versatile binding properties, and offer too little information density to fold unambiguously. This review covers the recent literature that seeks to create an improved platform to support laboratory Darwinism, one based on an artificially expanded genetic information system (AEGIS) that adds independently replicating nucleotide "letters" to the evolving "alphabet".

Mineral-Organic Interactions in Prebiotic Synthesis. The Discontinuous Synthesis Model for the Formation of RNA in Naturally Complex Geological Environments.
Steven A. Benner, Hyo-Joong Kim, and Elisa Biondi
Nucl. Acids & Mol. Bio.35, Springer 31-83 (2018) https://doi.org/10.1007/978-3-319-93584-3_3
<Abstract>

A common criticism of "prebiotic chemistry research" is that it is done with starting materials that are too pure, in experiments that are too directed, to get results that are too scripted, under conditions that could never have existed on Earth. Planetary scientists in particular remark that these experiments often arise simply because a chemist has a "cool idea" and then pursues it without considering external factors, especially geological and planetary context. A growing literature addresses this criticism and is reviewed here. We assume a model where RNA emerged spontaneously from a prebiotic environment on early Earth, giving the planet its first access to Darwinism. This "RNA First Hypothesis" is not driven by the intrinsic prebiotic accessibility; quite the contrary, RNA is a "prebiotic chemist's nightmare." However, by assuming models for the accretion of the Earth, the formation of the Moon, and the acquisition of Earth's "late veneer," a reasonable geological model can be envisioned to deliver the organic precursors needed to form the nucleobases and ribose of RNA. A geological model having an environment with dry arid land under a carbon dioxide atmosphere receiving effluent from serpentinizing igneous rocks allows their conversion to nucleosides and nucleoside phosphates. Mineral elements including boron and molybdenum prevent organic material from devolving to form "tars" along the way. And dehydration and activation allows the formation of oligomeric RNA that can be stabilized by adsorption on available minerals.

"Skinny" and "Fat" DNA: Two New Double Helices
Hoshika S, Singh I, Switzer C, Molt RW Jr, Leal NA, Kim MJ, Kim MS, Kim HJ, Georgiadis MM, Benner SA
J. Am. Chem. Soc. (2018) Sep 19;140(37):11655-11660. doi: 10.1021/jacs.8b05042. Epub 2018 Sep 10
<Abstract>

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide
Isha Singh, Roberto Laos, Shuichi Hoshika, Steven A. Benner, and Millie M. Georgiadis
Nucl. Acids Res.46(15) 7977-7988 (2018) doi: 10.1093/nar/gky552
<Abstract>

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2- amino-8-(1-beta-D-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro- 1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

Multiplexed isothermal amplification based diagnostic platform to detect Zika, chikungunya, and dengue-1.
Yaren, O., Alto, B. W., Bradley, K. M., Moussatche, P., Benner, S. A.
J. Vis. Exp., JoVE (2018) 133: e57051, DOI:10.3791/57051
<Abstract>

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time. Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to "approved" reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel. This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Molybdenum(VI)-catalyzed rearrangement of prebiotic carbohydrates in formamide, a candidate prebiotic solvent.
Ziegler, E.W., Kim, H.-J., Benner, S.A.
Astrobiology (2018) 18(9): 1159-70, DOI:10.1089/ast.2017.1742
<Abstract>

It has been four decades since formamide was first suggested to perform roles as a precursor and/or a solvent in prebiotic chemistry. However, little work has sought to integrate formamide into larger prebiotic schemes that might create prebiotic RNA, often proposed to have been the first Darwinian biopolymer. Here, we report that formamide can be used as a solvent to perform the Bílik reaction, which uses molybdenum(VI) oxo species as catalysts at near-neutral pH to rearrange branched carbohydrates to give linear carbohydrates; the branched carbohydrates are produced from formaldehyde (HCHO) in alkaline mixtures containing borate, whereas the linear carbohydrates are the precursors needed for ribonucleosides and ribonucleotides. Under conditions wherein the Bílik reaction does this rearrangement, carbohydrate reaction products do not require stabilization by borate. These results, therefore, connect aqueous and formamide-based processes for the prebiotic formation of RNA components. Based on data from Hadean zircons that show that the mantle of the early Earth was near the fayalite-quartz-magnetite fugacity, molybdenum in its 6+ oxidation state was likely available in the Hadean. Together, these allow us to conjecture a process that delivers ribonucleosides and ribonucleotides from hydrogen cyanide and HCHO from a Hadean atmosphere on a Hadean geosphere, without needing precisely timed transitions from one solvent system to the other.

Nucleoside analogs to manage sequence divergence in nucleic acid amplification and SNP detection.
Yang, Z., Kim, H.-J., Le, J., McLendon, C., Bradley, K.M., Kim, M.-S., Hutter, D., Hoshika, S., Yaren, O., Benner, S.A.
Nucl. Acids Res. (2018) 46(12): 5902-10,DOI:10.1093/nar/gky392
<Abstract>

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.

The Challenge of Synthetic Biology. Synthetic Darwinism and the Aperiodic Crystal Structure.
Karalkar, N.B. and Benner, S.A.
Curr. Op. Chem Biol. (2018) 46C:188-195, DOI:10.1016/j.cbpa.2018.07.008
<Abstract>

'Grand Challenges' offer ways to discover flaws in existing theory without first needing to guess what those flaws are. Our grand challenge here is to reproduce the Darwinism of terran biology, but on molecular platforms different from standard DNA. Access to Darwinism distinguishes the living from the non-living state. However, theory suggests that any biopolymer able to support Darwinism must (a) be able to form Schrödinger's 'aperiodic crystal', where different molecular components pack into a single crystal lattice, and (b) have a polyelectrolyte backbone. In 1953, the descriptive biology of Watson and Crick suggested DNA met Schrödinger's criertion, forming a linear crystal with geometrically similar building blocks supported on a polyelectrolye backbone. At the center of genetics were nucleobase pairs that fit into that crystal lattice by having both size complementarity and hydrogen bonding complementarity to enforce a constant geometry. This review covers experiments that show that by adhering to these two structural rules, the aperiodic crystal structure is maintained in DNA having 6 (or more) components. Further, this molecular system is shown to support Darwinism. Together with a deeper understanding of the role played in crystal formation by the poly-charged backbone and the intervening scaffolding, these results define how we might search for Darwinism, and therefore life, on Mars, Europa, Enceladus, and other watery lagoons in our Solar System.

Optimization of cationic (Q)-paper for detection of arboviruses in infected mosquitoes
Glushakova, L.G., Alto, B.W., Kim, M.S., Wiggins, K., Eastmond, B., Moussatche, P., Burkett-Cadena, N.D., and Benner, S.A.
J Virol Methods, Elsevier (2018) 261:71-9, DOI:10.1016/j.jviromet.2018.08.004
<Abstract>

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper’s binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated. Sixteen variations of Q-paper were prepared with modifications of the derivatizing reagents and derivatization temperature. The binding capacities of these variations were determined first with 1,3,5-benzenetricarboxylic (BTCA), then viral RNA (purified or in infected mosquito samples) was used for validation. For this, samples with Zika (ZIKV) and chikungunya (CHIKV) RNA or virus-infected Aedes aegypti mosquito bodies were applied to sixteen Q-paper variants. Washing the paper samples with water versus elution with aqueous salt (1 M) gave samples that were analyzed for viral RNA by a PCR-based direct Luminex hybridization assay. The comparison ranked the Q-paper binding capacities from the lowest to the highest. The Q-paper with the highest RNA binding capability was further validated with ZIKV- and CHIKV-infected mosquito saliva.

Detection of Mosquito-borne Arboviruses by twenty two multiplexed xMAP Luminex Arrays Panel
Glushakova, L.G., Alto, B.W., Bradley, A., Benner, S.A.
Res. & Rev. J. Microbiol. Biotechnol., Research & Reviews (2018) 7(2):36-40
<Abstract>

A 22-fold multiplexed Luminex panel to detect medically important arboviruses was previously reported targeting small RNAs produced in vitro. Two synthetic biology innovations made this level of multiplexing possible. We asked if multiplexing was robust when nine targets were added from the Flaviviridae, Togaviridae and Bunyaviridae families. Assay sensitivities were 10, 80, and 270 genome-equivalents for California encephalitis, chikungunya and Murray Valley encephalitis viruses, indicating the robustness of multiplexing using these innovations. Amounts of these viruses in a single infected mosquito are typically higher. Further, the panel identified Murray Valley encephalitis virus in a single infected-mosquito pooled with (5-50) uninfected mosquitoes.

Other Genetic Alphabets
Campos, L., Benner, S.A.
JoDS (2018) 4:10 Dec

Prebiotic Plausibility and Networks of Paradox-Resolving Independent Models
Benner, S.A.
Nat. Commun., Nature (2018) 9(1):5173, DOI:10.1038/s41467-018-07274-y
<Abstract>

The plausibility of any model in science comes from the extent of its interconnections to other models that are grounded in different premises and reasoning. Focusing research on paradoxes in those models, logic whereby they appear to generate unacceptable conclusions from seemingly indisputable premises, helps find those interconnections.

Butylacrylate-nucleobase Conjugates as Targets for Two-step Redox Labeling of DNA with an Osmium Tetroxide Complex
Havranova-VidlakovA, P., Spacek, J., Vítova, L., Hermanova, M., Dadova, J., Raindlova, V., Havran, L., et al.
Electroanalysis, Wiley (2018) 30(2), 371-377. DOI: 10.1002/elan.201700702
<Abstract>

Modification of nucleic acids with osmium tetroxide reagents (Os,L, such as OsO₄,2,2'-bipyridine, Os,bpy) has been applied in redox DNA labeling, in probing DNA structure as well as in studies of DNA interactions with other molecules. In natural DNA, primarily thymine residues form adducts with the Os,bpy in a structure selective manner. In this paper we introduce a new two-step technique of DNA modification with the electroactive Os,bpy, consisting in enzymatic construction of DNA bearing butyl acrylate (BA) moieties attached to uracil at C5 or to 7-deaza adenine at C7, followed by chemical modification of a reactive C=C double bond in the acrylate residue. We demonstrate a facile modification of the BA conjugates in both single- and double-stranded (ds) DNA under conditions when modification within the nucleobase rings in ds DNA is hindered. Various DNA-Os,bpy adducts can easily be analyzed electrochemically and distinguished by different redox potentials. The two-step procedure appears to be applicable in osmium redox labelling of ds DNA.

Metabolic reconstructions identify plant 3-methylglutaconyl-CoA hydratase that is crucial for branched-chain amino acid catabolism in mitochondria
Latimer S., Li Y., Nguyen T.TH., Soubeyrand E., Fatihi A., Elowsky C.G., Block A., Pichersky E., Basset G.J.
Plant J. (2018) 95 (2), 358-370. doi: 10.1111/tpj.13955
<Abstract>

The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant-prokaryote comparative genomics detected candidates for 3-methylglutaconyl-CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non-homologous N-terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein-fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3-hydroxymethylglutaryl-CoA into 3-methylglutaconyl-CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark-induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3-methylglutaconyl-CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.

Biophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2?a]-1,3,5-triazin-4(8H)one (P)
Xiaoyu Wang, Shuichi Hoshika, Raymond J. Peterson, Myong-Jung Kim, Steven A. Benner, and Jason D. Kahn
ACS Synthetic Biology, American Chemical Society (2017) DOI: 10.1021/acssynbio.6b00224
<Abstract>

Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°₃₇) for oligonucleotide hybridization were extracted. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair observed for C:O⁶-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodynamics of oligonucleotides containing Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.

Adsorption of RNA on mineral surfaces and mineral precipitates
Elisa Biondi, Yoshihiro Furukawa, Jun Kawai, and Steven A. Benner
Beilstein J. Org. Chem., Beilstein Institute (2017) 13, 393-404
<Abstract>

The prebiotic significance of laboratory experiments that study the interactions between oligomeric RNA and mineral species is difficult to know. Natural exemplars of specific minerals can differ widely depending on their provenance. While laboratory-generated samples of synthetic minerals can have controlled compositions, they are often viewed as "unnatural". Here, we show how trends in the interaction of RNA with natural mineral specimens, synthetic mineral specimens, and co-precipitated pairs of synthetic minerals, can make a persuasive case that the observed interactions reflect the composition of the minerals themselves, rather than their being simply examples of large molecules associating nonspecifically with large surfaces. Using this approach, we have discovered Periodic Table trends in the binding of oligomeric RNA to alkaline earth carbonate minerals and alkaline earth sulfate minerals, where those trends are the same when measured in natural and synthetic minerals. They are also validated by comparison of co-precipitated synthetic minerals. We also show differential binding of RNA to polymorphic forms of calcium carbonate, and the stabilization of bound RNA on aragonite. These have relevance to the prebiotic stabilization of RNA, where such carbonate minerals are expected to have been abundant, as they appear to be today on Mars.

Tautomeric equilibria of iso-guanine and related purine analogs
Nilesh B. Karalkar, Kshitij Khare, Robert Molt, and Steven A. Benner
Nuc. Nuc. Nuc. acids, Taylor & Francis Group (2017) Apr 3;36(4):256-274. doi: 10.1080/15257770.2016.1268694
<Abstract>

Nucleobase pairs in DNA match hydrogen-bond donor and acceptor groups on the nucleobases. However, these can adopt more than one tautomeric form, and can consequently pair with nucleobases other than their canonical complements, possibly a source of natural mutation. These issues are now being revisited by synthetic biologists increasing the number of replicable pairs in DNA by exploiting unnatural hydrogen bonding patterns, where tautomerism can also create mutation. Here, we combine spectroscopic measurements on methylated analogs of isoguanine tautomers and tautomeric mixtures with statistical analyses to a set of isoguanine analogs, the complement of isocytosine, the 5th and 6th "letters" in DNA.

Prebiotic stereoselective synthesis of purine and noncanonical pyrimidine nucleotide from nucleobases and phosphorylated carbohydrates
Hyo-Joong Kim, Steven A. Benner
Proc. Natl. Acad. Sci. USA (2017) October, 114 (43) 11315-11320. https://doi.org/10.1073/pnas.1710778114
<Abstract>

According to a current "RNA first" model for the origin of life, RNA emerged in some form on early Earth to become the first biopolymer to support Darwinism here. Threose nucleic acid (TNA) and other polyelectrolytes are also considered as the possible first Darwinian biopolymer(s). This model is being developed by research pursuing a "Discontinuous Synthesis Model" (DSM) for the formation of RNA and/or TNA from precursor molecules that might have been available on early Earth from prebiotic reactions, with the goal of making the model less discontinuous. In general, this is done by examining the reactivity of isolated products from proposed steps that generate those products, with increasing complexity of the reaction mixtures in the proposed mineralogical environments. Here, we report that adenine, diaminopurine, and hypoxanthine nucleoside phosphates and a noncanonical pyrimidine nucleoside (zebularine) phosphate can be formed from the direct coupling reaction of cyclic carbohydrate phosphates with the free nucleobases. The reaction is stereoselective, giving only the β-anomer of the nucleotides within detectable limits. For purines, the coupling is also regioselective, giving the N-9 nucleotide for adenine as a major product. In the DSM, phosphorylated carbohydrates are presumed to have been available via reactions explored previously [Krishnamurthy R, Guntha S, Eschenmoser A (2000) Angew Chem Int Ed 39:2281-2285], while nucleobases are presumed to have been available from hydrogen cyanide and other nitrogenous species formed in Earth's primitive atmosphere.

Detecting Darwinism from Molecules in the Enceladus Plumes, Jupiter's Moons, and Other Planetary Water Lagoons
Steven A. Benner
Astrobiology17 840-851 (2017) DOI: 10.1089/ast.2016.1611
<Abstract>

To the astrobiologist, Enceladus offers easy access to a potential subsurface biosphere via the intermediacy of a plume of water emerging directly into space. A direct question follows: If we were to collect a sample of this plume, what in that sample, through its presence or its absence, would suggest the presence and/or absence of life in this exotic locale? This question is, of course, relevant for life detection in any aqueous lagoon that we might be able to sample. This manuscript reviews physical chemical constraints that must be met by a genetic polymer for it to support Darwinism, a process believed to be required for a chemical system to generate properties that we value in biology. We propose that the most important of these is a repeating backbone charge; a Darwinian genetic biopolymer must be a "polyelectrolyte". Relevant to mission design, such biopolymers are especially easy to recover and concentrate from aqueous mixtures for detection, simply by washing the aqueous mixtures across a polycharged support. Several device architectures are described to ensure that, once captured, the biopolymer meets two other requirements for Darwinism, homochirality and a small building block "alphabet." This approach is compared and contrasted with alternative biomolecule detection approaches that seek homochirality and constrained alphabets in non-encoded biopolymers. This discussion is set within a model for the history of the terran biosphere, identifying points in that natural history where these alternative approaches would have failed to detect terran life. Key Words: Enceladus-Life detection-Europa-Icy moon- Biosignatures-Polyelectrolyte theory of the gene. Astrobiology 17, 840-851.

Expanded Genetic Alphabets. Managing Nucleotides that Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural Nucleotides
Winiger, C.B., Shaw, R.W., Kim, M.J., Moses, J.D., Matsuura, M.F. and Benner, S.A.
ACS Synthetic Biology, American Chemical Society (2017) 55(51):15816-20, DOI:10.1002/anie.201608001
<Abstract>

2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs. In analogous in vivo experiments, E. coli lacking dKTP/dXTP cannot rescue chloramphenicol resistance from a plasmid containing two adjacent K:X pairs. These studies identify bacteria able to serve as selection environments for engineering cells that replicate AEGIS pairs that lack forms that are Watson-Crick complementary to any natural nucleobase.

Uniting Natural History with the Molecular Sciences. The Ultimate Multidisciplinarity
Benner, S.A.
Acc. Chem. Res. (2017) 50(3):498-502, DOI:10.1021/acs.accounts.6b00496
<Abstract>

Life and the Earth have coevolved over the past four billion years to deliver a rich diversity of biological structure, from biomolecules to macrophysiology. One grand challenge seeks to interconnect these structures, in ways acceptable to both natural historians and physical scientists, to give an interconnecting web of models and experiments to create a planetary understanding of the phenomenon that we call "life". The molecular scientist wants experiments; the natural historian wants reference to Darwinian fitness. Paleogenetics offers both.

Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.
Chen, F., Zhang, Y., Daugherty, A.B., Yang, Z.Y., Shaw, R., Dong, M.X., Lutz, S. and Benner, S.A.
PLOS One, Public Library of Science (2017) 12(3): e0174163, DOI:10.1371/journal.pone.0174163
<Abstract>

One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1'-b-d-2'-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1'-b-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya
Yaren, O., Alto, B.W., Gangodkar, P.V., Ranade, S.R., Patil, K.N., Bradley, K.M., Yang, Z., Phadke, N., Benner, S.A
BMC Infect. Dis., BioMed Central Ltd. (2017) 17(1):293, DOI:10.1186/s12879-017-2382-0
<Abstract>

Background: Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives.

Methods: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration.

Results: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India.

Conclusions: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

Detection of chikungunya viral RNA in mosquito bodies on cationic (Q) paper based on innovations in synthetic biology
Glushakova, L.G., Alto, B.W., Kim, M.S., Bradley, A., Yaren, O., Benner, S.A.
J Virol Methods, Elsevier (2017) 246:104-11, DOI:10.1016/j.jviromet.2017.04.013
<Abstract>

Chikungunya virus (CHIKV) represents a growing and global concern for public health that needs inexpensive and convenient methods to collect mosquitoes as potential carriers so that they can be preserved, stored and transported for later and/or remote analysis. Reported here is a cellulose-based paper, derivatized with quaternary ammonium groups ("Q-paper") that meets these needs. In a series of tests, infected mosquito bodies were squashed directly on Q-paper. Aqueous ammonia was then added on the mosquito bodies to release viral RNA that adsorbed on the cationic surface via electrostatic interactions. The samples were then stored (frozen) or transported. For analysis, the CHIKV nucleic acids were eluted from the Q-paper and PCR amplified in a workflow, previously developed, that also exploited two nucleic acid innovations, ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS). The amplicons were then analyzed by a Luminex hybridization assay. This procedure detected CHIKV RNA, if present, in each infected mosquito sample, but not in non-infected counterparts or ddH2O samples washes, with testing one week or ten months after sample collection.

Structure and biophysics for a six letter DNA alphabet that includes imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K).
Singh, I., Kim, M.J., Molt, R., Benner, S. A., Georgiadis, M.
ACS Synthetic Biology, American Chemical Society (2017) 6(11):2118-29, DOI:10.1021/acssynbio.7b00150
<Abstract>

A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson–Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host–guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs.

Nucleic Acid Crystallization and Phase Determination Facilitated by Selenium Functionalization
X. Chen, C. Chen, J. Gan, W. Zhang, O. O. Gerlits, J. Salon, J. Caton-Williams, S. Jiang, H. Liu and Z. Huang
Acta Crystallogr., Wiley (2017) A73, a186
<Abstract>

Selenium atom-specific functionalization can offer nucleic acids with many unique and novel properties (such as facilitated crystallization and phase determination) without significant perturbation of 3D structures of nucleic acids and their protein complexes. Nucleic acids possess not only the ability to store genetic information and participate in transcription and translation, but also the capacity to adopt well-defined 3D structures, which can be readily adjusted to meet various functional needs (such as catalysis and therapeutics). Although the importance of numerous nucleic acids in catalysis, gene expression, protein binding and therapeutics has been acknowledged by the entire scientific society, current understanding of nucleic acid-protein functions and structures is still limited, especially highresolution structures. This novel Se-atom-specific functionalization will provide important tools to investigate nucleic acid structure/folding, recognition and catalysis, to study nucleic acids and their protein interactions, to improve biochemical and biophysical properties of nucleic acids, and to explore potential nucleic acid therapeutics and diagnostics. Our presentation will focus on the most recent selenium-atom functionalization of nucleic acids and their potential applications in 3D structure-and-function studies and anticancer therapeutics in molecular medicine.

Label-free detection of canonical DNA bases, uracil and 5-methylcytosine in DNA oligonucleotides using linear sweep voltammetry at a pyrolytic graphite electrode
Spacek, J., Danhel, A., Hason, S., & Fojta, M.
Electrochem. commun., Elsevier (2017) 82, 34-38. DOI: 10.1016/j.elecom.2017.07.013
<Abstract>

An innovative approach to label-free voltammetric analysis of DNA at a pyrolytic graphite electrode (PGE) within a broad range of potentials (from - 2.0 to + 1.6 V) in an acetate buffer (pH 5) is presented. Using specifically designed DNA nonamers, we demonstrate not only anodic oxidation, but for the first time also cathodic reduction of nucleobases at the PGE. In addition, products of irreversible oxidation/reduction of the parent bases are shown to yield analytically useful, base-specific cathodic/anodic signals, making it possible to distinguish between the canonical bases (adenine, cytosine, guanine and thymine), uracil (U) and 5-methylcytosine (mC) in DNA. Furthermore, selective electrochemical "switching off" of the redox signals specific to certain nucleobases is presented as a way to resolve overlapping signals. Similarly, newly reported signals corresponding to electrochemically transformed bases can be “switched on” under specific conditions. This approach can be utilized for fast and facile simultaneous label-free analysis of bases in DNA, including mC and U, and to uncover overlapping signals. This significantly extends the possible applications of PGE in DNA research and (bio)sensor development.

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase
Li Y., Martin J.R., Aldama G.A., Fernadez D.E. and Cline K.
Plant Physiol, Oxford (2017) 173(4): 2121-2137. doi: 10.1104/pp.17.00012.
<Abstract>

Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40.

Isothermal titration calorimetry uncovers substrate promiscuity of bicupin oxalate oxidase from Ceriporiopsis subvermispora
Rana H, Moussatche P, Rocha LS, Abdellaoui S, Minteer SD, Moomaw EW
Biochem. Biophys. Rep., Elsevier (2016) 5:396-400 doi:10.1016/j.bbrep.2016.01.016
<Abstract>

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzymecatalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (?Happ). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6- sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.

Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase
Mariko F. Matsuura, Ryan W. Shaw, Jennifer D. Moses, Hyo-Joong Kim, Myong-Jung Kim, Myong-Sang Kim, Shuichi Hoshika, Nilesh Karalkar, and Steven A. Benner
ACS Synthetic Biology, American Chemical Society (2016) 5 (3), pp 234-240 DOI: 10.1021/acssynbio.5b00172
<Abstract>

One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3- (1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin- 4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System
Karalkar NB, Leal NA, Kim MS, Bradley KM, Benner SA
ACS Synthetic Biology, American Chemical Society (2016) doi: 10.1021/acssynbio.5b00276
<Abstract>

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ~92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ~86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.

Allosteric inhibitors of Coxsackie virus A24 RNA polymerase
CH Schein, D Rowold, KH Choi
Bioorg. Med. Chem. (2016) Volume 24, Issue 4, 15 February 2016, Pages 570-577

Cross-Reactivity Among Peanut and Tree Nut Allergens
SJ Maleki, H Cheng, JC Wolf, SS Teuber, C Schein, CC Grimm
J Allergy Clin Immunol, American Academy of Allergy, Asthma & Immunology (2016) 137 (2), AB237

Alternative Watson-Crick Synthetic Genetic Systems
Steven A. Benner, Nilesh B. Karalkar, Shuichi Hoshika, Roberto Laos, Ryan W. Shaw, Mariko Matsuura, Diego Fajardo, and Patricia Moussatche
Cold Spring Harb Perspect Biol, Cold Spring Harbor Laboratory Press (2016) doi: 10.1101/cshperspect.a023770
<Abstract>

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution
Zhang, L, Yang, Z, Trinh, TL, Teng, I-T, Wang, S, Bradley, KM, Hoshika, S, Wu, Q, Cansiz, S, Rowold, DJ, McLendon, C, Kim, M-S, Wu, Y, Cui, C, Liu, Y, Hou, W, Stewart, K, Wan, S, Liu, C, Benner, SA, Tan, W
Angew. Chem. Int. Ed.55 (2016) doi: 10.1002/anie.201605058
<Abstract>

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a sixletter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a]- [1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selectionâ€“counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus
Ozlem Yaren, Lyudmyla G. Glushakova, Kevin M. Bradley, Shuichi Hoshika,Steven A. Benner
J Virol Methods(236), Elsevier 54-61 (2016) doi:10.1016/j.jviromet.2016.07.008
<Abstract>

This paper combines two advances to detect MERS-CoV, the causative agent of Middle East Respiratory Syndrome, that have emerged over the past few years from the new field of "synthetic biology". Both are based on an older concept, where molecular beacons are used as the downstream detection of viral RNA in biological mixtures followed by reverse transcription PCR amplification. The first advance exploits the artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four found in standard DNA and RNA (xNA); AEGIS nucleotides pair orthogonally to the A:T and G:C pairs. Placing AEGIS components in the stems of molecular beacons is shown to lower noise by preventing unwanted stem invasion by adventitious natural xNA. This should improve the signal-to-noise ratio of molecular beacons operating in complex biological mixtures. The second advance introduces a nicking enzyme that allows a single target molecule to activate more than one beacon, allowing "signal amplification". Combining these technologies in primers with components of a self-avoiding molecular recognition system (SAMRS), we detect 50 copies of MERS-CoV RNA in a multiplexed respiratory virus panel by generating fluorescence signal visible to human eye and/or camera.

A norovirus detection architecture based on isothermal amplification and expanded genetic systems
Ozlem Yaren, Kevin M. Bradley, Patricia Moussatche, Shuichi Hoshika, Zunyi Yang,Shu Zhu, Stephanie M. Karst, Steven A. Benner
J Virol Methods(237), Elsevier 64-71 (2016) doi: 10.1016/j.jviromet.2016.08.012
<Abstract>

Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel.

Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary Implications
Christian B. Winiger, Myong-Jung Kim, Shuichi Hoshika, Ryan W. Shaw, Jennifer D. Moses, Mariko F. Matsuura, Dietlind L. Gerloff, and Steven A. Benner
Biochemistry55(28), ACS 3847-3850 (2016) DOI: 10.1021/acs.biochem.6b00533
<Abstract>

In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen
Biondi E, Lane JD, Das D, Dasgupta S, Piccirilli JA, Hoshika S, Bradley KM, Krantz BA, Benner SA
Nucl. Acids Res. (2016) Oct 3. pii: gkw890. PubMed PMID: 27701076
<Abstract>

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ~35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.

Crystal structures of deprotonated nucleobases from an expanded DNA alphabet
Mariko F. Matsuura, Hyo-Joong Kim, Daisuke Takahashi, Khalil A. Abboud and Steven A. Benner
Structural Chemistry, Acta Crystallographica (2016) C72, 952-959. doi: 10.1107/S2053229616017071
<Abstract>

Reported here is the crystal structure of a heterocycle that implements a donor–donor–acceptor hydrogen-bonding pattern, as found in the Z component [6-amino-5-nitropyridin-2(1H)-one] of an artificially expanded genetic information system (AEGIS). AEGIS is a new form of DNA from synthetic biology that has six replicable nucleotides, rather than the four found in natural DNA. Remarkably, Z crystallizes from water as a 1:1 complex of its neutral and deprotonated forms, and forms a ‘skinny’ pyrimidine–pyrimidine pair in this structure. The pair resembles the known intercalated cytosine pair. The formation of the same pair in two different salts, namely poly[[aqua(µ6-2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ido)sodium]–6-amino-5-nitropyridin-2(1H)-one–water (1/1/1)], denoted Z-Sod, {[Na(C5H4N3O3)(H2O)]·C5H5N3O3·H2O}n, and ammonium 2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ide–6-amino-5-nitropyridin-2(1H)-one–water (1/1/1), denoted Z-Am, NH4+·C5H4N3O3·C5H5-N3O3·H2O, under two different crystallization conditions suggests that the pair is especially stable. Implications of this structure for the use of this heterocycle in artificial DNA are discussed.

Opal Absorbs and Stabilizes RNA - A Hierarchy of Prebiotic Silica Minerals
Biondi, E.; Howell, L.; Benner, S.A.
SynLett (2016) 27, A-E
<Abstract>

A widely held 'RNA first' model proposes that RNA gave organic matter on Earth its first access to Darwinism. Such a proposal, which requires a mechanism to generate RNA from a prebiotic 'soup', must also manage the intrinsic instability of any RNA so formed. Here, we show that silicon dioxide (silica, SiO2), in the form of synthetic opal, adsorbs and stabilizes RNA from aqueous solution. The extent of absorption on fully amorphous silica is less, as is the extent of adsorption on the surface of crystalline quartz. We show that the RNA adsorbed on opal is considerably more stable than the same RNA molecule free in aqueous solution at pH 9.5. This provides a mechanism by which any RNA formed in a prebiotic environment could have been concentrated and stabilized so that it could have later participated in the first Darwinian biology.

A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System
Mariko F. Matsuura, Christian B. Winiger, Ryan W. Shaw, Myong-Jung Kim, Myong-Sang Kim, Ashley B. Daugherty, Fei Chen, Patricia Moussatche, Jennifer D. Moses, Stefan Lutz, and Steven A. Benner
ACS Synthetic Biology, American Chemical Society (2016) DOI: 10.1021/acssynbio.6b00228
<Abstract>

ABSTRACT: Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-D-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-D-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-D-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

Unusual Hydrogen Bonding Patterns and the Role of the Backbone in Nucleic Acid Information Transfer
Steven A. Benner
ACS Cent Sci.(2), American Chemical Society 882-884 (2016) DOI: 10.1021/acscentsci.6b00344

Evaporite Borate-Containing Mineral Ensembles Make Phosphate Available and Regiospecifically Phosphorylate Ribonucleosides: Borate as a Multifaceted Problem Solver in Prebiotic Chemistry
Kim, H.J., Furukawa, Y., Kakegawa, T., Bita, A., Scorei, R. and Benner, S.A.
Angew. Chem. Int. Ed. (2016) 55(51):15816-20, DOI:10.1002/anie.201608001
<Abstract>

RNA is currently thought to have been the first biopolymer to support Darwinian natural selection on Earth. However, the phosphate esters in RNA and its precursors, and the many sites at which phosphorylation might occur in ribonucleosides under conditions that make it possible, challenge prebiotic chemists. Moreover, free inorganic phosphate may have been scarce on early Earth owing to its sequestration by calcium in the unreactive mineral hydroxyapatite. Herein, it is shown that these problems can be mitigated by a particular geological environment that contains borate, magnesium, sulfate, calcium, and phosphate in evaporite deposits. Actual geological environments, reproduced here, show that Mg²⁺ and borate sequester phosphate from calcium to form the mineral lüneburgite. Ribonucleosides stabilized by borate mobilize borate and phosphate from lüneburgite, and are then regiospecifically phosphorylated by the mineral. Thus, in addition to guiding carbohydrate pre-metabolism, borate minerals in evaporite geoorganic contexts offer a solution to the phosphate problem in the "RNA first" model for the origins of life.

A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division
Li Y., Liu D, López-Paz C., Olson B.J., Umen J.G.
eLife, Howard Hughes Medical Institute, Max Planck Society, and Wellcome Trust (2016) 25; 5: e10767. doi: 10.7554/eLife.10767
<Abstract>

Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control.

Evolution of functional six-nucleotide DNA
Zhang, L., Yang, Z., Sefah, K., Bradley, K. M., Hoshika, S., Kim, M-J,. Kim, H-J., Zhu., Jimenez, E., Cansiz, S., Teng, I-T., Champanhac, C, McLendon, C., Liu, C., Zhang, W., Gerloff, D. L., Huang, Z., Tan, W., Benner, S. A.
J. Am. Chem. Soc. (2015) DOI: 10.1021/jacs.5b02251
<Abstract>

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5- nitro-3-(1'-B-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)-imidazo- [1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson?Crick geometry. These were added to a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

Transcription, Reverse Transcription, and Analysis of RNA Containing Artificial Genetic Components
Nicole A. Leal, Hyo-Joong Kim, Shuichi Hoshika, Myong-Jung Kim, Matthew A. Carrigan, and Steven A. Benner
ACS Synthetic Biology, American Chemical Society (2015) Apr 17;4(4):407-13. doi: 10.1021/sb500268n
<Abstract>

Expanding the synthetic biology of artificially expanded genetic information systems (AEGIS) requires tools to make and analyze RNA molecules having added nucleotide "letters". We report here the development of T7 RNA polymerase and reverse transcriptase to catalyze transcription and reverse transcription of xNA (DNA or RNA) having two complementary AEGIS nucleobases, 6-amino-5-nitropyridin-2-one (trivially, Z) and 2-aminoimidazo[1,2a]-1,3,5-triazin-4(8H)-one (trivially, P). We also report MALDI mass spectrometry and HPLC-based analyses for oligomeric GACUZP six-letter RNA and the use of ribonuclease (RNase) A and T1 RNase as enzymatic tools for the sequence-specific degradation of GACUZP RNA. We then applied these tools to analyze the GACUZP and GACTZP products of polymerases and reverse transcriptases (respectively) made from DNA and RNA templates. In addition to advancing this 6-letter AEGIS toward the biosynthesis of proteins containing additional amino acids, these experiments provided new insights into the biophysics of DNA.

Pooled assembly of marine metagenomic datasets: enriching annotation through chimerism
Jonathan D. Magasin and Dietlind L. Gerloff
Bioinformatics (2015) 31:311-317
<Abstract>

Motivation: Despite advances in high-throughput sequencing, marine metagenomic samples remain largely opaque. A typical sample contains billions of microbial organisms from thousands of genomes and quadrillions of DNA base pairs. Its derived metagenomic dataset underrepresents this complexity by orders of magnitude because of the sparseness and shortness of sequencing reads. Read shortness and sequencing errors pose a major challenge to accurate species and functional annotation. This includes distinguishing known from novel species. Often the majority of reads cannot be annotated and thus cannot help our interpretation of the sample.
Results: Here, we demonstrate quantitatively how careful assembly of marine metagenomic reads within, but also across, datasets can alleviate this problem. For 10 simulated datasets, each with species complexity modeled on a real counterpart, chimerism remained within the same species for most contigs (97%). For 42 real pyrosequencing ('454') datasets, assembly increased the proportion of annotated reads, and even more so when datasets were pooled, by on average 1.6% (max 6.6%) for species, 9.0% (max 28.7%) for Pfam protein domains and 9.4% (max 22.9%) for PANTHER gene families. Our results outline exciting prospects for data sharing in the metagenomics community. While chimeric sequences should be avoided in other areas of metagenomics (e.g. biodiversity analyses), conservative pooled assembly is advantageous for annotation specificity and sensitivity. Intriguingly, our experiment also found potentia

3D topology of the SMC2/SMC4 sub-complex from chicken condensin I revealed by cross-linking and molecular modeling
Barysz H, Kim JH, Chen ZA, Hudson DF, Rappsilber J, Gerloff DL, Earnshaw WC
Open Biology, The Royal Society Publishing (2015) 5:150005
<Abstract>

SMC proteins are essential components of three protein complexes that are important for chromosome structure and function. The cohesin complex holds replicated sister chromatids together, whereas the condensin complex has an essential role in mitotic chromosome architecture. Both are involved in interphase genome organisation. SMC-containing complexes are large (>650 kDa for condensin) and contain long anti-parallel coiled-coils. They are thus difficult subjects for conventional crystallographic and electron cryomicroscopic studies. Here we have used amino acid-selective cross-linking and mass spectrometry (CLMS) combined with structure prediction to develop a full-length molecular draft 3D-structure of the SMC2/SMC4 dimeric backbone of chicken condensin. We assembled homology-based molecular models of the globular heads and hinges with the lengthy coiled-coils modelled in fragments, using numerous high-confidence cross-links and accounting for potential irregularity. Our experiments reveal that isolated condensin complexes can exist with their coiled-coil segments closely apposed to one another along their lengths and define the relative spatial alignment of the two anti-parallel coils. The centres of the coiled-coils can also approach one another closely in situ in mitotic chromosomes. In addition to revealing structural information, our cross-linking data suggest that both H2A and H4 may have roles in condensin interactions with chromatin.

Formation of Two Homo-chromophoric H-Aggregates in DNA-Assembled Alternating Dye Stacks
C.B. Winiger; Dr. S.M. Langenegger; Prof. Dr. G. Calzaferri; Prof. Dr. R. Haner
Angew. Chem. Int. Ed.(54) 3643-3647 (2015) DOI:10.1002/anie.201410041

Structural basis for a six nucleotide genetic alphabet
Georgiadis, M. M., Singh, I., Kellett, W. F., Hoshika, S., Benner, S. A. Richards, N. G. J.
J. Am. Chem. Soc. (2015) DOI: 10.1021/jacs.5b3482
<Abstract>

Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]- 1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard "edge on" Watson?Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Angstrom wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.

Sequence specificity for uridylylation of the viral peptide linked to the genome (VPg) of enteroviruses
Catherine H Schein, Mengyi Ye, Aniko V Paul, M Steven Oberste, Nora Chapman, Dmitri V Filippov, Kyung Choi
Virology, Elsevier (2015) 484:80-85

Inositol hexakisphosphate analogs and uses thereof
TC Savidge, P Urvil, D Nauduri, N Oezguen, C Schein, W Braun
US Patent, United States Patent Office (2015) Patent No. 9,200,015

Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA
Lyudmyla G. Glushakova, Nidhi Sharma, Shuichi Hoshika, Andrea C. Bradley, Kevin M. Bradley, Zunyi Yang, Steven A. Benner
Anal Biochem, Elsevier (2015) Nov 15;489:62-72. doi: 10.1016/j.ab.2015.08.015
<Abstract>

Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-ml sample.

High-throughput multiplexed xMAP Luminex array panel for detection of twenty two medically important mosquito-borne arboviruses based on innovations in synthetic biology
Lyudmyla G. Glushakova, Andrea Bradley, Kevin M. Bradley, Barry W. Alto, Shuichi Hoshika, Daniel Hutter, Nidhi Sharma, Zunyi Yang, Myong-Jung Kim, Steven A. Benner
J Virol Methods214, Elsevier 60-74 (2015) doi: 10.1016/j.jviromet.2015.01.003
<Abstract>

Mosquito-borne arboviruses are emerging world-wide as important human and animal pathogens. This makes assays for their accurate and rapid identification essential for public health, epidemiological, ecological studies. Over the past decade, many mono- and multiplexed assays targeting arboviruses nucleic acids have been reported. None has become established for the routine identification of multiple viruses in a "single tube" setting. With increasing multiplexing, the detection of viral RNAs is complicated by noise, false positives and negatives. In this study, an assay was developed that avoids these problems by combining two new kinds of nucleic acids emerging from the field of synthetic biology. The first is a "self-avoiding molecular recognition system" (SAMRS), which enables high levels of multiplexing. The second is an "artificially expanded genetic information system" (AEGIS), which enables clean PCR amplification in nested PCR formats. A conversion technology was used to place AEGIS component into amplicon, improving their efficiency of hybridization on Luminex beads. When Luminex "liquid microarrays" are exploited for downstream detection, this combination supports single-tube PCR amplification assays that can identify 22 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, Orthobunyavirus. The assay differentiates between closely-related viruses, as dengue, West Nile, Japanese encephalitis, and the California serological group. The performance and the sensitivity of the assay were evaluated with dengue viruses and infected mosquitoes; as few as 6-10 dengue virions can be detected in a single mosquito.

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair
Armando R. Hernandez, Yaming Shao, Shuichi Hoshika, Zunyi Yang, Sandip A. Shelke, Julien Herrou, Hyo-Joong Kim, Myong-Jung Kim, Joseph A. Piccirilli, and Steven A. Benner
Angew. Chem. Int. Ed.54 9853-9856 (2015) doi: 10.1002/anie.201504731
<Abstract>

As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6- amino-5-nitro-2(1H)-pyridone and 2-aminoimidazo[ 1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNAmolecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nm affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.

Restriction enzymes cleave DNA immobilized on micron-sized diamond crystallites
Yaren O, Benner SA
Diamond Relat. Mater., Elsevier (2015) 52, 18-24
<Abstract>

Because diamonds have strongly bonded networks of carbon atoms, they offer the potential to support DNA-targeted analysis in architectures that require very stable DNA immobilization with very low DNA leakage. Further, their non-porous structures should allow diamond-immobilized DNA to easily gain access to enzymes in bulk solution. As part of our work to develop a molecular biology tool kit to transform immobilized DNA, we asked whether diamond-immobilized DNA could be cleaved by sequence-specific restriction endonucleases, despite the large sizes of those enzymes, the potential for "steric" obstruction from the diamond surface, and the possibility that the diamond surface might inactivate those enzymes. We report here that both standard and "nicking" restriction endonucleases cut diamond-immobilized single-stranded DNA, after it forms a duplex with a complementary strand of DNA delivered from solution. As a somewhat surprising result, we also discovered that restriction enzymes could cleave a fraction of the immobilized duplex DNA even if the complementary strand came not from solution, but rather from a separate diamond crystallite. This cleavage did not result from a failure of the attachment linkage that allowed the diffusion of leaked DNA through bulk solvent. Rather, the cleavage required physical proximity between crystallites, as confirmed by transmission electron microscopy. These results add to the tools that can use diamond-immobilized DNA, as well as define practical constraints on assay architectures where diamond-immobilized DNA is presumed to be isolated from other diamond-immobilized DNA particles.

Helicase Dependent Isothermal Amplification of DNA and RNA using Self-Avoiding Molecular Recognition Systems
Zunyi Yang, Chris McLendon, Daniel Hutter, Kevin M. Bradley, Shuichi Hoshika, Carole Frye, and Steven A. Benner
ChemBioChem (2015) June 15; 16(9): 1365-1370. doi:10.1002/cbic.201500135.
<Abstract>

Assays that target DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low resource environments, requiring equipment and power that may not be available in these environments. However, isothermal procedures that avoid thermal cycling are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts to give clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, something difficult to achieve with HDA using standard primers. This shows that SAMRS-HDA is a more versatile approach than standard HDA with a broader applicability for xNA-targeted diagnostics and research.

Solubility of Polyethers in Hydrocarbons at Low Temperatures. A Model for Potential Genetic Backbones on Warm Titans
Christopher McLendon, F. Jeffrey Opalko, Heshan I. Illangkoon, and Steven A. Benner
Astrobiology15(3) (2015) DOI: 10.1089/ast.2014.1212
<Abstract>

Ethers are proposed here as the repeating backbone linking units in linear genetic biopolymers that might support Darwinian evolution in hydrocarbon oceans. Hydrocarbon oceans are found in our own solar system as methane mixtures on Titan. They may be found as mixtures of higher alkanes (propane, for example) on warmer hydrocarbon-rich planets in exosolar systems ("warm Titans"). We report studies on the solubility of several short polyethers in propane over its liquid range (from 85 to 231 K, or - 188°C to - 42°C). These show that polyethers are reasonably soluble in propane at temperatures down to ca. 200 K. However, their solubilities drop dramatically at still lower temperatures and become immeasurably low below 170 K, still well above the ~95 K in Titan's oceans. Assuming that a liquid phase is essential for any living system, and genetic biopolymers must dissolve in that biosolvent to support Darwinism, these data suggest that we must look elsewhere to identify linear biopolymers that might support genetics in Titan's surface oceans. However, genetic molecules wih polyether backbones may be suitable to support life in hydrocarbon oceans on warm Titans, where abundant organics and environments lacking corrosive water might make it easier for life to originate.

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component
Li Y., Singhal R., Taylor W. Isaiah., McMinn H.P., Chua X.Y., Cline K., and Fernandez D. E.
Plant J. (2015) 84: 647-658, DOI: 10.1111/tpj.13028
<Abstract>

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid-based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co-immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma-exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.

RNA structural analysis by enzymatic digestion
Biondi E., Burke D.H.
Methods Mol Biol, Springer (2014) 1086:41-52

Protein Domains of Unknown Function Are Essential in Bacteria
Norman F. Goodacre, Dietlind L. Gerloff, Peter Uetz
mBio, American Society for Microbiology (2014) 5(1):e00744-13
<Abstract>

More than 20% of all protein domains are currently annotated as "domains of unknown function" (DUFs). About 2,700 DUFs are found in bacteria compared with just over 1,500 in eukaryotes. Over 800 DUFs are shared between bacteria and eukaryotes, and about 300 of these are also present in archaea. A total of 2,786 bacterial Pfam domains even occur in animals, including 320 DUFs. Evolutionary conservation suggests that many of these DUFs are important. Here we show that 355 essential proteins in 16 model bacterial species contain 238 DUFs, most of which represent single-domain proteins, clearly establishing the biological essentiality of DUFs. We suggest that experimental research should focus on conserved and essential DUFs (eDUFs) for functional analysis given their important function and wide taxonomic distribution, including bacterial pathogens.

Applications of the Complementarity Plot in error detection and structure validation of proteins
Sankar Basu, Dhananjay Bhattacharyya and Rahul Banerjee
Indian J. Biochem. Biophys., NISCAIR (2014) 51 (June) (Accepted, In press)

Membrane Interaction and Functional Plasticity of Inositol Polyphosphate 5-Phosphatases
Werner Braun and Catherine H. Schein
Structure (2014) http://dx.doi.org/10.1016/j.str.2014.04.008

Ribonucleosides for an Artificially Expanded Genetic Information System
Hyo-Joong Kim, Nicole A. Leal, Shuichi Hoshika, Steven A. Benner
J. Org. Chem. (2014) 79 (7), pp 3194-3199
<Abstract>

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2?-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson?Crick complement,imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA.

OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS) to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands
Kevin M. Bradley and Steven A. Benner
Beilstein J. Org. Chem., Beilstein Institute (2014) 10, 1826-1833, doi:10.3762/bjoc.10.192
<Abstract>

Synthetic biologists wishing to self-assemble large DNA (L-DNA) constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson–Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS), and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting") software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology.

Recombinase-Based Isothermal Amplification of Nucleic Acids with Self-Avoiding Molecular Recognition Systems (SAMRS)
Nidhi Sharma, Shuichi Hoshika, Daniel Hutter, Kevin M. Bradley, and Steven A. Benner
ChemBioChem (2014) DOI: 10.1002/cbic.201402250
<Abstract>

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

Influence of perylenediimide-pyrene supramolecular interactions on the stability of DNA-based hybrids: Importance of electrostatic complementarity
C.B. Winiger; Dr. S.M. Langenegger; Dr. O. Khorev; Prof. Dr. R. Haner
Beilstein J. Org. Chem.(10), Beilstein Institute 1589-1595 (2014) DOI:10.1002/anie.201407968

Long Distance EET in Light-Harvesting Supramolecular Polymers
C.B. Winiger; Dr. S. Li; Dr. G.R. Kumar; Dr. S.M. Langenegger; Prof. Dr. R. Haner
Angew. Chem. Int. Ed.(53) 13609-13613 (2014) DOI:10.1002/anie.201407968

DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides. Frontiers in Microbiology
Laos, R., Thomson, J. M., & Benner, S. A.
Frontiers in Microbiology (2014) 5, 565. http://doi.org/10.3389/fmicb.2014.00565
<Abstract>

DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic "alphabet." This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution.

Hominids adapted to metabolize ethanol long before human-directed fermentation
Matthew A. Carrigan, Oleg Uryasev, Carole B. Frye, Blair L. Eckman, Candace R. Myers, Thomas D. Hurley, and Steven A. Benner
Proc. Natl. Acad. Sci. USA112(2) 458-463 (2014) doi: 10.1073/pnas.1404167111
<Abstract>

Paleogenetics is an emerging field that resurrects ancestral proteins from now-extinct organisms to test, in the laboratory, models of protein function based on natural history and Darwinian evolution. Here, we resurrect digestive alcohol dehydrogenases (ADH4) from our primate ancestors to explore the history of primate–ethanol interactions. The evolving catalytic properties of these resurrected enzymes show that our ape ancestors gained a digestive dehydrogenase enzyme capable of metabolizing ethanol near the time that they began using the forest floor, about 10 million y ago. The ADH4 enzyme in our more ancient and arboreal ancestors did not efficiently oxidize ethanol. This change suggests that exposure to dietary sources of ethanol increased in hominids during the early stages of our adaptation to a terrestrial lifestyle. Because fruit collected from the forest floor is expected to contain higher concentrations of fermenting yeast and ethanol than similar fruits hanging on trees, this transition may also be the first time our ancestors were exposed to (and adapted to) substantial amounts of dietary ethanol.

Asymmetric alkyne addition to aldehydes catalyzed by Schiff bases made from 1,1'-bi-2-naphthol and chiral benzylic amines
Cen Chen, Qingfei Huang, Sheng Zou, Lei Wang, Bao Luan, Jin Zhu, Qiwei Wang, Lin Pu
Tetrahedron Asymmetry, Elsevier (2014) 25:199-201, DOI:10.1016/j.tetasy.2013.12.013

MaGnET: Malaria Genome Exploration Tool
Sharman JL and Gerloff DL
Bioinformatics (2013) 2350-2352
<Abstract>

Summary: The Malaria Genome Exploration Tool (MaGnET) is a software tool enabling intuitive 'exploration-style' visualization of functional genomics data relating to the malaria parasite, Plasmodium falciparum. MaGnET provides innovative integrated graphic displays for different datasets, including genomic location of genes, mRNA expression data, protein–protein interactions and more. Any selection of genes to explore made by the user is easily carried over between the different viewers for different datasets, and can be changed interactively at any point (without returning to a search).

Availability and Implementation: Free online use (Java Web Start) or download (Java application archive and MySQL database; requires local MySQL installation) at http://malariagenomeexplorer.org
Contact: [email protected] or [email protected]

Potent Inhibition of HIV-1 Reverse Transcriptase and Replication by Nonpseudoknot, "UCAA-motif" RNA Aptamers
Whatley A.S., Ditzler M.A., Lange M.J., Biondi E., Sawyer A.W., Chang J.L., Franken J.D., Burke D.H.
Mol Ther Nucleic Acids, Nature (2013) 2:e71

Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants
Moomaw EW, Hoffer E, Moussatche P, Salerno JC, Grant M, et al.
PLOS One, Public Library of Science (2013) 8(3): e57933
<Abstract>

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.

Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns
Laos R, Shaw R, Leal NA, Gaucher E, Benner S.
Biochemistry, ACS (2013) 52, 5288-5294
<Abstract>

Artificial genetic systems have been developed by synthetic biologists over the past two decades to include additional nucleotides that form additional nucleobase pairs independent of the standard T:A and C:G pairs. Their use in various tools to detect and analyze DNA and RNA requires polymerases that synthesize duplex DNA containing unnatural base pairs. This is especially true for nested polymerase chain reaction (PCR), which has been shown to dramatically lower noise in multiplexed nested PCR if nonstandard nucleotides are used in their external primers. We report here the results of a directed evolution experiment seeking variants of Taq DNA polymerase that can support the nested PCR amplification with external primers containing two particular nonstandard nucleotides, 2-amino-8-(1'-B-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (trivially called P) that pairs with 6-amino-5-nitro-3-(1'-B-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z). Variants emerging from the directed evolution experiments were shown to pause less when challenged in vitro to incorporate dZTP opposite P in a template. Interestingly, several sites involved in the adaptation of Taq polymerases in the laboratory were also found to have displayed "heterotachy" (different rates of change) in their natural history, suggesting that these sites were involved in an adaptive change in natural polymerase evolution. Also remarkably, the polymerases evolved to be less able to incorporate dPTP opposite Z in the template, something that was not selected. In addition to being useful in certain assay architectures, this result underscores the general rule in directed evolution that "you get what you select for".

Conversion strategy using an expanded genetic alphabet to assay nucleic acids
Yang, Z., Durante, M., Glushakova, L., Sharma, N., Leal, N., Bradley, K., Chen, F., Benner, S. A.
Anal. Chem. (2013) 85(9):4705-12
<Abstract>

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

The "strong" RNA world hypothesis. Fifty years old.
Neveu, Mark; Kim, Hyo-Joong; Benner, Steven A
Astrobiology13(4) (2013) DOI: 10.1089/ast.2012.0868
<Abstract>

This year marks the 50th anniversary of a proposal by Alex Rich that RNA, as a single biopolymer acting in two capacities, might have supported both genetics and catalysis at the origin of life. We review here both published and previously unreported experimental data that provide new perspectives on this old proposal. The new data include evidence that, in the presence of borate, small amounts of carbohydrates can fix large amounts of formaldehyde that are expected in an environment rich in carbon dioxide. Further, we consider other species, including arsenate, arsenite, phosphite, and germanate, that might replace phosphate as linkers in genetic biopolymers. While linkages involving these oxyanions are judged to be too unstable to support genetics on Earth, we consider the possibility that they might do so in colder semi-aqueous environments more exotic than those found on Earth, where cosolvents such as ammonia might prevent freezing at temperatures well below 273 K. These include the ammonia-water environments that are possibly present at low temperatures beneath the surface of Titan, Saturn’s largest moon.

Models and Standards of Proof in Cross-Disciplinary Science: The Case of Arsenic DNA
Steven A. Benner, William Bains, and Sara Seager
Astrobiology13(5) (2013) DOI: 10.1089/ast.2012.0954

Nucleic Acid Polymerases. Chapter 7: Engineered DNA Polymerases
Roberto Laos, Ryan W. Shaw, Steven A. Benner
Nucl. Acids & Mol. Bio., Springer (2013)
<Abstract>

This chapter reviews the methods used to generate variants of DNA polymerases that have improved ability, in particular to accept unnatural nucleotides, focusing especially on in vitro and directed evolution methods. Several natural families of DNA polymerases have independently evolved for millions of years to accept their natural nucleotide substrates with high fidelity and the ability to exclude closely related structures, such as ribonucleoside derivatives. However, polymerases that can accept unnatural nucleotide substrates would have many applications in biotechnology. Directed evolution may be an efficient method to produce new DNA polymerases capable to do so. Directed evolution relies on methods to create a library of sequence diverse polymerases starting with a gene for a parent polymerase. These methods are reviewed here, as well as examples of their application to produce variant polymerases. An evolutionary rationalization is offered to explain some mutations produced by directed evolution experiments.

Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme
Biondi E., Poudyal R.R., Forgy J.C., Sawyer A.W., Maxwell A.W., Burke D.H
Nucl. Acids Res. (2013) 41(5):3327-38

Assessment of 3D models for allergen research
Power, T.D, Ivanciuc, O., Schein, C.H. and Braun, W.
Proteins: Struct., Funct., Bioinf., Wiley (2013) 81 (4):545-554

A general synthetic method toward uridylylated picornavirus VPg proteins
Heden van Noort, G.J., Schein, C.H., Overkleeft, H.S., Marel, G.A., Filippov, D.V.
J. Pept. Sci., Wiley (2013) 19 (6):333-336

SARAMA: A Standalone Suite of Programs for the Complementarity Plot-A Graphical Structure Validation Tool for Proteins
Sankar Basu, Dhananjay Bhattacharyya and Rahul Banerjee
J. Bioinf. Intell. Control, American Scientific Publishers (2013) 2 (4): 321-323

polDNAmelt: Local Melting Within Polymeric DNA-An Improved Method and Its Applications
Sankar Basu and Dhananjay Bhattacharyya
J. Bioinf. Intell. Control, American Scientific Publishers (2013) 2 (4): 316-320

DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid-Targeted Lu-177-Radionuclide Tumor Therapy in Mice
Dr. C. Mueller; Dr. H. Struthers; C. Winiger; Dr. K. Zhernosekov; Prof. Dr. R. Schibli
J. Nucl. Med.(54), Society of Nuclear Medicine and Molecular Imaging 124-131 (2013) DOI: 10.2967/jnumed.112.107235

Engineered DNA Polymerases
K. Murakami and M.A. Trakselis (eds.)
Nucleic Acid Polymerases, Nucleic Acids and Molecular Biology, Springer-Verlag Berlin Heidelberg (2013)

Use of codon models in molecular dating and functional analysis
Steven A. Benner
Codon Evolution, ed. Gina M Cannarozzi, Adrian Scheider, Oxford University Press 133-144 (2012)

Synthesis and Properties of 5-Cyano-Substituted Nucleoside Analog with a Donor-Donor-Acceptor Hydrogen-Bonding Pattern
Hyo-Joong Kim, Fei Chen, and Steven A. Benner
J. Org. Chem. (2012)
<Abstract>

6-Aminopyridin-2-ones form Watson-Crick pairs with complementary purine analogues to add a third nucleobase pair to DNA and RNA, if an electron-withdrawing group at position 5 slows oxidation and epimerization. In previous work with a nucleoside analogue trivially named dZ, the electron withdrawing unit was a nitro group. Here, we describe an analogue of dZ (cyano-dZ) having a cyano group instead of a nitro group, including its synthesis, pKa, rates of acid-catalyzed epimerization, and enzymatic incorporation.

The Natural History of Class I Primate Alcohol Dehydrogenases Includes Gene Duplication, Gene Loss, and Gene Conversion
Matthew A. Carrigan, Oleg Uryasev, Ross P. Davis, LanMin Zhai, Thomas D. Hurley, Steven A. Benner
PLOS One7(7), Public Library of Science (2012)

Non-genomic progesterone signalling and its non-canonical receptor
Moussatche P, Lyons TJ
Biochem Soc Trans (2012) Feb;40(1):200-4
<Abstract>

The steroid hormone progesterone regulates many critical aspects of vertebrate physiology. The nuclear receptor for progesterone functions as a ligand-activated transcription factor, directly regulating gene expression. This type of signalling is referred to as the 'genomic' pathway. Nevertheless, progesterone also stimulates rapid physiological effects that are independent of transcription. This pathway, termed 'non-genomic', is mediated by the mPRs (membrane progesterone receptors). These mPRs belong to a larger class of membrane receptors called PAQRs (progestin and adipoQ receptors), which include receptors for adiponectin in vertebrates and osmotin in fungi. mPRs have been shown to activate inhibitory G-proteins, suggesting that they act as GPCRs (G-protein-coupled receptors). However, PAQRs do not resemble GPCRs with respect to topology or conserved sequence motifs. Instead, they more closely resemble proteins in the alkaline ceramidase family and they may possess enzymatic activity. In the present paper, we highlight the evidence in support of each model and what is currently known for PAQR signal transduction of this non-canonical receptor.

A small ribozyme with dual-site kinase activity
Biondi E., Maxwell A.W.R., and Burke D.H.
Nucl. Acids Res. (2012) 40(15):7528-40

Separating and analyzing sulfur-containing RNAs with organomercury gels
Biondi E, Burke DH.
Methods Mol Biol, Springer (2012) 2012;883:111-20
<Abstract>

Polyacrylamide gel electrophoresis is a widely used technique for RNA analysis and purification. The polyacrylamide matrix is highly versatile for chemical derivitization, enabling facile exploitation of thio-mercury chemistry without the need of tedious manipulations and/or expensive coupling reagents, which often give low yields and side products. Here, we describe the use of [(N-acryloylamino)phenyl]mercuric chloride in three-layered polyacrylamide gels to detect, separate, quantify, and analyze sulfur-containing RNAs.

Structure-Based Redesign of an Edema Toxin Inhibitor
Chen, D, Ma, L., Kanalas, J.J., Gao, J, Pawlik J, Estrella-Jimenez, JE, Walter M.A., Peterson, JW, Gilbertson S.R. and Schein C.H.
Bioorg. Med. Chem. (2012) 20, 368-376 PMCID: PMC3251925

Simplifying complex sequence information: a PCP-consensus protein binds antibodies against all four Dengue serotypes
Bowen, D.M., Lewis, J.A., Lu, W. and Schein, C.H.
Vaccine, Elsevier (2012) 30(42), 6081-6087

Physicochemical property consensus sequences for functional analysis, design of multivalent antigens and targeted antivirals
Schein, C.H., Bowen, D.M., Lewis, J.A., Choi, K., Paul, A., van der Heden van Noort, G.J., Lu, W. and Filippov, D.V.
BMC Bioinformatics (2012) 13(Suppl 13):S9.

Ara h 1 structure is retained after roasting and is important for enhanced binding to IgE
Nesbit, J.B., Hurlburt, B.K., Schein, C.H., Cheng, H. and Maleki, S.J.
Mol. Nutr. Food Res., Wiley (2012) 56, 1-9

Pharmacophore Selection and Redesign of Non-nucleotide Inhibitors of Anthrax Edema Factor
Schein C.H., Chen D, Ma L, Kanalas JJ, Gao J, Jimenez ME, Sower LE, Walter MA, Gilbertson SR, Peterson JW
Toxins, MDPI (2012) 4(11):1288-1300

Self Complementarity within proteins: bridging the gap between binding and folding
Sankar Basu, Dhananjay Bhattacharyya and Rahul Banerjee
Biophys. J., AP Cell Press (2012) 102 (11): 2605-2614

Synthesis and incorporation into oligodeoxynucleotides of carbocyclic exo-amino nucleosides
Yaren O, Rothlisberger P, Leumann CJ
Synthesis, Thieme (2012) 7, 1011-1025
<Abstract>

The preparation of a series of carbocyclic exo-amino nucleosides from selected primary aromatic amines in both the C1' α- and β-epimeric form as well as the corresponding building blocks for DNA synthesis is described. These nucleosides were incorporated into oligodeoxynucleotides and their base-pairing properties with natural bases and, in part, with themselves investigated. The results obtained confirm that such nucleoside analogues engage in specific interactions with natural nucleotides in DNA duplexes. In addition they can form self-pairs that match or exceed the stability of Watson-Crick base-pairs. Thus, carbocyclic exo-amino nucleosides can be taken into consideration in the design of novel base-pairs for the extension of the genetic alphabet, or for other applications in biotechnology.

The maize tapetum employs diverse mechanisms to synthesize and store proteins and flavonoids and transfer them to the pollen surface
Li Y., Suen D.F., Huang C.Y., Kung, S.Y. and Huang A.H.C.
Plant Physiol, Oxford (2012) 158: 1548-1561, DOI: 10.1104/pp.111.189241
<Abstract>

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.

Recognition of an expanded genetic alphabet by type-II restriction endonucleases and their application to analyze polymerase fidelity.
Chen, F; Yang, ZY; Yan, M; Alvarado, JB; Wang, G; Benner, SA
Nucl. Acids Res.39(9) 3949-3961 (2011)
<Abstract>

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1'-?-d-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-?-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor-donor-acceptor and acceptor-acceptor-donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells.

Characterization of Ceriporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris
Moussatche, P; Angerhofer, A; Imaram, W; Hoffer, E; Uberto, K; Brooks, C; Bruce, C; Sledge, D; Richards, NGJ; Moomaw, EW
Arch. Biochem. Biophys. (2011) May 1;509(1):100-7. Epub 2011 Mar 2
<Abstract>

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.

Synthetic Biology, Tinkering Biology, and Artificial Biology: A Perspective from Chemistry
Benner, SA; Chen, F; Yang, ZY
Chemical Synthetic Biology, ed. Pier Luigi Luisi and Cristiano Chiarabelli, Wiley 69-106 (2011)

Setting the Stage: The History, Chemistry, and Geobiology behind RNA
Benner, SA; Kim, HJ; Yang, ZY
RNA Worlds: From Life's Origins to Diversity in Gene Regulation, ed. John F. Atkins, Raymond F. Gesteland, Thomas R. Cech, Cold Spring Harbor Laboratory Press 7-19 (2011)

Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System
Yang, Z; Chen, F; Alvarado, JB; Benner, SA
J. Am. Chem. Soc.133(38) 15105-15112 (2011) dx.doi.org/10.1021/ja204910n
<Abstract>

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a sixletter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Phylogenetic and preliminary phenotypic analysis of yeast PAQR receptors: potential antifungal targets
Villa NY, Moussatche P, Chamberlin SG, Kumar A, Lyons TJ
J Mol Evol (2011) Oct;73(3-4):134-52. Epub 2011 Oct 19.
<Abstract>

Proteins belonging to the Progestin and AdipoQ Receptor (PAQR) superfamily of membrane bound receptors are ubiquitously found in fungi. Nearly, all fungi possess two evolutionarily distinct paralogs of PAQR protein, which we have called the PQRA and PQRB subtypes. In the model fungus Saccharomyces cerevisiae, these subtypes are represented by the Izh2p and Izh3p proteins, respectively. S. cerevisiae also possesses two additional PQRA-type receptors called Izh1p and Izh4p that are restricted to other species within the "Saccharomyces complex". Izh2p has been the subject of several recent investigations and is of particular interest because it regulates fungal growth in response to proteins produced by plants and, as such, represents a new paradigm for interspecies communication. We demonstrate that IZH2 and IZH3 gene dosage affects resistance to polyene antifungal drugs. Moreover, we provide additional evidence that Izh2p and Izh3p negatively regulate fungal filamentation. These data suggest that agonists of these receptors might make antifungal therapeutics, either by inhibiting fungal development or by sensitizing fungi to the toxic effects of current antifungal therapies. This is particularly relevant for pathogenic fungi such as Candida glabrata that are closely related to S. cerevisiae and contain the same complement of PAQR receptors.

Computationally Predicted IgE Epitopes of Walnut Allergens Contribute to Cross-Reactivity with peanuts
Maleki, S.J., Teuber, S.S, Cheng H, Chen D, Comstock, S.S., Ruan, S., and Schein, C.H.
Allergy, Wiley (2011) 66(12), 1522-1529. PMCID: PMC3203311

Mapping the distribution of packing topologies within protein interiors shows predominant preference for specific packing motifs
Sankar Basu, Dhananjay Bhattacharyya and Rahul Banerjee
BMC Bioinformatics (2011) 12:195

An SNP-Based Linkage Map for Zebrafish Reveals Sex Determination Loci
Bradley KM, Breyer JP, Melville DB, Broman KW, Knapik EW, Smith JR
G3, Genetics Society of America (2011) Jun 1;1(1):3-9
<Abstract>

A surprising diversity of mechanisms controls sex determination of vertebrate organisms, even among closely related species. Both genetic and temperature-dependent systems of sex determination have been described in teleost fish. In the common zebrafish model organism, heteromorphic sex chromosomes are not observed, and the potential role of a genetic component of sex determination remains largely unknown. Here we report a genome-wide linkage study of sex determination in zebrafish using a novel SNP genetic map. We identified loci on zebrafish chromosomes 5 (LOD score 7.9) and 16 (LOD score 9.3) governing sex determination as a complex trait, rather than as an XY or ZW genetic system. Each of these loci contains a prominent candidate gene with a conserved role in sex determination across additional species that suggest potential mechanisms of sex determination in zebrafish. The chromosome 5 locus harbors dmrt1, a key gene in sex determination from fruit flies to humans; mutation of the human DMRT1 ortholog is a cause of complete sex reversal of XY individuals. The chromosome 16 locus harbors cyp21a2; mutation of the human CYP21A2 ortholog is one of the more common causes of pseudohermaphroditism. Mutation detection at each of these candidate genes within the zebrafish cross identified hypomorphic variants on the female-associated allele of each locus. The two loci together accounted for 16% of variance of the trait. Interacting environmental cues are likely to be an additional important component of sex determination in zebrafish.

A Parallel Screen for the Discovery of Novel DNA Base Pairs
Yaren O, Mosimann M, Leumann CJ
Angew. Chem. Int. Ed. (2011) 50, 1935-1938. PMID: 21328674
<Abstract>

A combinatorial assay was developed for screening a library of aromatic heterocyclic amines for their propensity to act as a complementary base Z in a DNA duplex. This assay may prove useful in speeding up the process of base-pair discovery for potential applications in biotechnology and synthetic biology.

Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups
Hutter, D; Kim, MJ; Karalkar, N; Leal, NA; Chen, F; Guggenheim, E; Visalakshi, V; Olejnik, J; Gordon, S; Benner, SA
Nuc. Nuc. Nuc. acids29(11), Taylor & Francis Group 879-895 (2010)
<Abstract>

Nucleoside triphosphates having a 3'-ONH(2) blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3'-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3'-ONH(2) group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3'-ONH(2) blocking group in "next generation sequencing."

Expanded Genetic Alphabets in the Polymerase Chain Reaction
Yang, ZY; Chen, F; Chamberlin, SG; Benner, SA
Angew. Chem. Int. Ed.49(1) 177-180 (2010)

Artificial Genetic Systems: Self-Avoiding DNA in PCR and Multiplexed PCR
Hoshika, S; Chen, F; Leal, NA; Benner, SA
Angew. Chem. Int. Ed.49(32) 5554-5557 (2010)

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection
Chen, F; Gaucher, EA; Leal, NA; Hutter, D; Havemann, SA; Govindarajan, S; Ortlund, EA; Benner, SA
Proc. Natl. Acad. Sci. USA107(5) 1948-1953 (2010)

Chemistry, Life, and the Search for Aliens
Benner, SA
Proc. SPIE7819(10) 1-12 (2010)
<Abstract>

While "life" may universally be a self-sustaining chemical system capable of Darwinian evolution, alien life may be quite different in its chemistry from the terran life that we know here on Earth. In this case, it will be difficult to recognize, especially if it has not advanced beyond the single cell life forms that have dominated much of the terran biosphere. This review summarizes what we might infer from general physical and chemical law about how such "weird" life might be structured, what solvents other than water it might inhabit, what genetic molecules it might contain, and what metabolism it might exploit.

Improving efficiencies of locus-specific DNA methylation assessment for bovine in vitro produced embryos
Wroclawska, E; Brant, JO; Yang, TP; Moore, K
Syst. Biol. Reprod. Med.56 96-105 (2010)

Interview with Steven Benner
Impey, C; Benner, SA
Talking about Life: Conversations on Astrobiology, ed. Chris Impey, Cambridge University Press 58-68 (2010)

Q&A: Life, synthetic biology and risk
Benner, SA
BMC Biology8(77) (2010) doi:10.1186/1741-7007-8-77

Synthetic biology, tinkering biology, and artificial biology. What are we learning?
Steven A. Benner, Zunyi Yang, and Fei Chen
Comptes Rendus Chimie, French Academy of Sciences (2010)
<Abstract>

While chemical theory cannot yet support an engineering vision that allows molecules, DNA sequences, and proteins to be interchangeable parts in arti?cial constructs without ‘‘tinkering’’, progress can be made in synthetic biology by pursuing challenges at the limits of existing theory. These force scientists across uncharted terrain where they must address unscripted problems where, if theory is inadequate, failure results. Thus, synthesis drives discovery and paradigm change in ways that analysis cannot. Further, if failures are analyzed, new theories emerge. Here, we illustrate this by synthesizing an artificial genetic system capable of Darwinian evolution, an ability theorized to be universal to life.

Convergent donor and acceptor substrate utilization among kinase ribozymes
Biondi E., Nickens D.G., Warren S., Saran D., and Burke D.H.
Nucl. Acids Res. (2010) 38(19):6785-95

2'-Deoxy-1-methylpseudocytidine, a stable analog of 2'-deoxy-5-methylisocytidine
Kim, HJ; Leal, NA; Benner, SA
Bioorg. Med. Chem.17(10) 3728-3732 (2009)
<Abstract>

2 '-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2'-deoxy-5-methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glycoside analog that is more stable and, in oligonucleotides, pairs with 2 '-deoxyisoguanosine, contributing to duplex stability about as much as a standard 2 '-deoxycytidine and 2 '-deoxyguanosine pair. (C) 2009 Elsevier Ltd. All rights reserved.

A Convenient Synthesis of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-Methyl-2'-Deoxypseudoisocytidine
Wellington, KW; Ooi, HC; Benner, SA
Nuc. Nuc. Nuc. acids28(4), Taylor & Francis Group 275-291 (2009)
<Abstract>

The syntheses of N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside and 1-methyl-2'-deoxypseudoisocytidine via Heck coupling are described. A survey of the attempts to use the Heck coupling to synthesize N,N'-dibenzyl-2,4-diaminopyrimidine-2'-deoxyribonucleoside is provided, indicating a remarkable diversity in outcome depending on the specific heterocyclic partner used.

Directed evolution of a filamentous fungus for thermotolerance
de Crecy, E; Jaronski, S; Lyons, B; Lyons, TJ; Keyhani, NO
BMC Biotechnology9 74 (2009)
<Abstract>

Background: Filamentous fungi are the most widely used eukaryotic biocatalysts in industrial and chemical applications. Consequently, there is tremendous interest in methodology that can use the power of genetics to develop strains with improved performance. For example, Metarhizium anisopliae is a broad host range entomopathogenic fungus currently under intensive investigation as a biologically based alternative to chemical pesticides. However, it use is limited by the relatively low tolerance of this species to abiotic stresses such as heat, with most strains displaying little to no growth between 35-37 degrees C. In this study, we used a newly developed automated continuous culture method called the Evolugator(TM) which takes advantage of a natural selection-adaptation strategy, to select for thermotolerant variants of M. anisopliae strain 2575 displaying robust growth at 37 degrees C. Results: Over a 4 month time course, 22 cycles of growth and dilution were used to select 2 thermotolerant variants of M. anisopliae. Both variants displayed robust growth at 36.5 degrees C, whereas only one was able to grow at 37 degrees C. Insect bioassays using Melanoplus sanguinipes (grasshoppers) were also performed to determine if thermotolerant variants of M. anisopliae retained entomopathogenicity. Assays confirmed that thermotolerant variants were, indeed, entomopathogenic, albeit with complex alterations in virulence parameters such as lethal dose responses (LD50) and median survival times (ST50). Conclusion: We report the experimental evolution of a filamentous fungus via the novel application of a powerful new continuous culture device. This is the first example of using continuous culture to select for complex phenotypes such as thermotolerance. Temperature adapted variants of the insect-pathogenic, filamentous fungus M. anisopliae were isolated and demonstrated to show vigorous growth at a temperature that is inhibitory for the parent strain. Insect virulence assays confirmed that pathogenicity can be retained during the selection process. In principle, this technology can be used to adapt filamentous fungi to virtually any environmental condition including abiotic stress and growth substrate utilization.

Signatures of a Shadow Biosphere
Davies, PCW; Benner, SA; Cleland, CE; Lineweaver, CH; McKay, CP; Wolfe-Simon, F
Astrobiology9(2) 241-249 (2009)
<Abstract>

Astrobiologists are aware that extraterrestrial life might differ from known life, and considerable thought has been given to possible signatures associated with weird forms of life on other planets. So far, however, very little attention has been paid to the possibility that our own planet might also host communities of weird life. If life arises readily in Earth-like conditions, as many astrobiologists contend, then it may well have formed many times on Earth itself, which raises the question whether one or more shadow biospheres have existed in the past or still exist today. In this paper, we discuss possible signatures of weird life and outline some simple strategies for seeking evidence of a shadow biosphere.

Antagonism of Human Adiponectin Receptors and Their Membrane Progesterone Receptor Paralogs by TNF alpha and a Ceramidase Inhibitor
Kupchak, BR; Garitaonandia, I; Villa, NY; Smith, JL; Lyons, TJ
Biochemistry48(24), ACS 5504-5506 (2009)
<Abstract>

The progestin and AdipoQ receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are nonclassical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1(S),2(R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNF alpha, a homologue of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.

Metal dependence of oxalate decarboxylase activity
Moomaw, EW; Angerhofer, A; Moussatche, P; Ozarowski, A; García-Rubio, I; Richards, NG
Biochemistry48(26), ACS 6116-6125 (2009)
<Abstract>

Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO2. These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.

Adiponectin identified as an agonist for PAQR3/RKTG using a yeast-based assay system
Garitaonandia, I; Smith, JL; Kupchak, BR; Lyons, TJ
J. Recept. Signal Transduction29(1) 67-73 (2009)
<Abstract>

The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered "orphan" receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.

Sphingolipids Function as Downstream Effectors of a Fungal PAQR
Villa, NY; Kupchak, BR; Garitaonandia, I; Smith, JL; Alonso, E; Alford, C; Cowart, LA; Hannun, YA; Lyons, TJ
Mol. Pharmacol.75(4) 866-875 (2009)
<Abstract>

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as D-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steady-state levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.

The challenges of sequencing by synthesis
Fuller, CW; Middendorf, LR; Benner, SA; Church, GM; Harris, T; Huang, XH; Jovanovich, SB; Nelson, JR; Schloss, JA; Schwartz, DC; Vezenov, DV
Nat. Biotechnol.27(11) 1013-1023 (2009)
<Abstract>

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $ 100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

Dissecting the regulation of yeast genes by the osmotin receptor
Kupchak, BR; Villa, NY; Kulemina, LV; Lyons, TJ
Biochem. Biophys. Res. Comm.374(2) 210-213 (2008)
<Abstract>

The Izh2p protein from Saccharomyces cerevisiae is a receptor for the plant antifungal protein, osmotin. Since Izh2p is conserved in fungi, understanding its biochemical function could inspire novel strategies for the prevention of fungal growth. However, it has been difficult to determine the exact role of Izh2p because it has pleiotropic effects on cellular biochemistry. Herein, we demonstrate that Izh2p negatively regulates functionally divergent genes through a CCCTC promoter motif. Moreover, we show that Izh2p-dependent promoters containing this motif are regulated by the Nrg1p/Nrg2p and Msn2p/Msn4p transcription factors. The fact that Izh2p can regulate gene expression through this widely dispersed element resents a reasonable explanation of its pleiotropy. The involvement of Nrg1p/Nrgp2 in Izh2p-dependent gene regulation also suggests a role for this receptor in regulating fungal differentiation in response to stimuli produced by plants. (C) 2008 Elsevier Inc. All rights reserved.

Design of a novel molecular beacon: modification of the stem with artificially genetic alphabet
Sheng, PP; Yang, ZY; Kim, YM; Wu, YR; Tan, WH; Benner, SA
Chem. Comm.(41), Royal Society of Chemistry 5128-5130 (2008)
<Abstract>

A molecular beacon that incorporates components of an artificially expanded genetic information system (AEGIS) in its stem is shown not to be opened by unwanted stem invasion by adventitious standard DNA; this should improve the "darkness" of the beacon in real-world applications.

The planetary biology of ascorbate and uric acid and their relationship with the epidemic of obesity and cardiovascular disease
Johnson, RJ; Gaucher, EA; Sautin, YY; Henderson, GN; Angerhofer, AJ; Benner, SA
Medical Hypotheses71(1) 22-31 (2008)
<Abstract>

Humans have relatively low plasma ascorbate levels and high serum uric acid levels compared to most mammals due to the presence of genetic mutations in L-gulonotactone oxidase and uricase, respectively. We review the major hypotheses for why these mutations may have occurred. In particular, we suggest that both mutations may have provided a survival advantage to early primates by helping maintain blood pressure during periods of dietary change and environmental stress. We further propose that these mutations have the inadvertent disadvantage of increasing our risk for hypertension and cardiovascular disease in today's society characterized by Western diet and increasing physical inactivity. Finally, we suggest that a "planetary biology" approach in which genetic changes are analyzed in relation to their biological action and historical context may provide the ideal approach towards understanding the biology of the past, present and future. (c) 2008 Elsevier Ltd. All rights reserved.

Self-Avoiding Molecular Recognition Systems (SAMRS)
Hoshika, S; Chen, F; Leal, NA; Benner, SA
Nucleic Acids Symp. Ser.52(1) 129-130 (2008)

Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure
Havemann, SA; Hoshika, S; Hutter, D; Benner, SA
Nuc. Nuc. Nuc. acids27(3), Taylor & Francis Group 261-278 (2008)
<Abstract>

In this article, we focus on the synthesis of aryl C-glycosides via Heck coupling. It is organized based on the type of structures used in the assembly of the C-glycosides (also called C-nucleosides) with the following subsections: pyrimidine C-nucleosides, purine C-nucleosides, and monocyclic, bicyclic, and tetracyclic C-nucleosides. The reagents and conditions used for conducting the Heck coupling reactions are discussed. The subsequent conversion of the Heck products to the corresponding target molecules and the application of the target molecules are also described.

Synthesis of pyrophosphates for in vitro selection of catalytic RNA molecules
Kim, HJ; Kim, MJ; Karalkar, N; Hutter, D; Benner, SA
Nuc. Nuc. Nuc. acids27, Taylor & Francis Group 43-56 (2008)

Heterologous expression of human mPR alpha, mPR beta and mPR gamma in yeast confirms their ability to function as membrane progesterone receptors
Smith, JL; Kupchak, BR; Garitaonandia, I; Hoang, LK; Maina, AS; Regalla, LM; Lyons, TJ
Steroids73(11) 1160-1173 (2008)
<Abstract>

The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPR alpha, mPR beta and mPR gamma, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPR alpha, mPR beta and mPR gamma can sense and respond to progesterone with EC50 values that are physiologically relevant. Agonist profiles also show that mPR alpha, mPR beta and mPR gamma are activated by ligands, such as 17 alpha-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs; have been renamed as mPR delta (PAQR6) and mPR epsilon (PAQR9). Additional characterization of mPR gamma and mPRa indicates that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs; as a novel class of G-protein coupled receptor. (c) 2008 Elsevier Inc. All rights reserved.

Synthetic Biology for Improved Personalized Medicine
Benner, SA; Hoshika, S; Sukeda, M; Hutter, D; Leal, NA; Yang, ZY; Chen, F
Nucleic Acids Symp. Ser.52(1) 243-244 (2008) doi: 10.1093/nass/nrn123
<Abstract>

Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk.

Paleotemperature trend for Precambrian life inferred from resurrected proteins
Gaucher, EA; Ganesh, O; Govindarajan, S
Nature (2007) In press

Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4'-thioribonucleosides
Hoshika, S; Minakawa, N; Shionoya, A; Imada, K; Ogawa, N; Matsuda, A
ChemBioChem8(17) 2133-2138 (2007)
<Abstract>

A detailed study of the modification pattern-RNAi activity relationships by using siRNAs that are modified with 4'-thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa-2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4'-thioribonucleoside unit on both ends of the sense strand and four residues on the 3'-end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4'-thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.

Computational reconstruction of ancestral genomic regions from evolutionarily conserved gene clusters
Danchin, EGJ; Gaucher, EA; Pontarotti, P
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 139-150 (2007)

Experimental resurrection of ancient biomolecules: gene synthesis, heterologous protein expression, and functional assays
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 153-163 (2007)

Ancestral sequence reconstruction as a tool to understand natural history and guide synthetic biology: realizing and extending the vision of Zuckerkandl and Pauling
Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 20-33 (2007)

A thermophilic last universal ancestor inferred from its estimated amino acid composition
Brooks, DJ; Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 200-207 (2007)

The resurrection of ribonucleases from mammals: from ecology to medicine
Sassi, SO; Benner, SA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 208-224 (2007)

The early days of paleogenetics: connecting molecules to the planet
Benner, SA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 3-19 (2007)

Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis
Svedruzic, D; Liu, Y; Reinhardt, LA; Wroclawska, E; Cleland, WW; Richards, NGJ
Arch. Biochem. Biophys.464(1) 36-47 (2007)
<Abstract>

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO2 and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies. (c) 2007 Elsevier Inc. All rights reserved.

Synthesis of a novel bicyclic nucleoside with a 3,7-anhydrooctofuranosyl skeleton
Kim, MJ; Chun, MW
Aust. J. Chem.60(4) 291-295 (2007)
<Abstract>

We have developed an efficient route for the synthesis of a novel bicyclic nucleoside using a key intermediate 13, which was prepared from 1,2: 5,6-di-O-isopropylidene-alpha-D-glucose. 1,2-Nucleophilic addition of aldehyde 8 with allyltrimethylsilane and intramolecular Williamson reaction were successfully achieved to synthesize an intermediate with a 3,7-anhydrooctofuranosyl skeleton.

Probing the mechanism of FET3 repression by Izh2p overexpression
Kupchak, BR; Garitaonandia, I; Villa, NY; Mullen, MB; Weaver, MG; Regalla, LA; Kendall, EA; Lyons, TJ
Biochim. Biophys. Acta1773(7) 1124-1132 (2007)
<Abstract>

We previously reported a role for the IZH2 gene product in metal ion metabolism. Subsequently, Izh2p was also identified as a member of the PAQR family of receptors and, more specifically, as the receptor for the plant protein osmotin. In this report, we investigate the effect of Izh2p on iron homeostasis. We show that overproduction of Izh2p prevents the iron-dependent induction of the Fet3p component of the high-affinity iron-uptake system and is deleterious for growth in iron-limited medium. We demonstrate that the effect of Izh2p requires cAMP-dependent kinase and AMP-dependent kinase and is not mediated by general inhibition of the Aft1p iron-responsive transcriptional activator. We also show that lzh2p-overproduction negatively regulates Nrg1p/Nrg2p- and Msn2p/Msn4p-dependent reporters. Furthermore, we show that the Nrg1p/Nrg2p and Msn2p/Msn4p pairs are epistatic to each other with respect to their effects on FET3 expression. Finally, we show that the mechanism by which PAQR receptors activate signal transduction pathways is likely to be conserved from yeast to humans. (C) 2007 Elsevier B.V. All rights reserved.

Transport and Storage of Metal Ions in Biology
Lyons, TJ; Eide, DJ
Biological Inorganic Chemistry: Structure and Reactivity, ed. I. Bertini, H. Gray, E. Stiefel, and J.S. Valentine, University Science Books 57-77 (2007)

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population
Settles, AM; Holding, DR; Tan, BC; Latshaw, SP; Liu, J; Suzuki, M; Li, L; O'Brien, BA; Fajardo, DS; Wroclawska, E; Tseung, CW; Lai, JS; Hunter, CT; Avigne, WT; Baier, J; Messing, J; Hannah, LC; Koch, KE; Becraft, PW; Larkins, BA; McCarty, DR
BMC Genomics8(1) 116-144 (2007)
<Abstract>

Background: Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations. Results: Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus- specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill. Conclusion: We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis
Ruhland, A; Leal, N; Kima, PE
Cell Microbiol.9(1) 84-96 (2007)
<Abstract>

Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NF kappa B and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NF kappa B signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

PduL is an evolutionarily distinct phosphotransacylase involved in B-12-dependent 1,2-propanediol degradation by Salmonella enterica serovar typhimurium LT2
Liu, Y; Leal, NA; Sampson, EM; Johnson, CLV; Havemann, GD; Bobik, TA
J. Bacteriol.189(5) 1589-1596 (2007)
<Abstract>

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B-12-dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His(8)) was purified, and its kinetic constants were determined: the V-max was 51.7 +/- 7.6 mu mol min(-1) mg(-1), and the K-m values for propionyl-PO42- and acetyl-PO42- were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.

In vivo expression of human ATP : cob(I)atamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
Erger, KE; Conlon, TJ; Leal, NA; Zori, R; Bobik, TA; Flotte, TR
J. Gene Med.9(6) 462-469 (2007)
<Abstract>

Background Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B-12, adenosylcobalamin (AdoCbl). AdoCbl is. a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Methods The human ATR cDNA was cloned into a recombinant adenoassociated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/B16 mice at a dose of 1 x 10(10) and 1 x 10(11), particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Results Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken P-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. Conclusions These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments. Copyright (C) 2007 John Wiley & Sons, Ltd.

The evolution of seminal ribonuclease: Pseudogene reactivation or multiple gene inactivation events?
Sassi, SO; Braun, EL; Benner, SA
Mol. Biol. Evol.24(4) 1012-1024 (2007)
<Abstract>

Two approaches, one novel, are applied to analyze the divergent evolution of ruminant seminal ribonucleases (RNases), paralogs of the well-known pancreatic RNases of mammals. Here, the goal was to identify periods of divergence of seminal RNase under functional constraints, periods of divergence as a pseudogene, and periods of divergence driven by positive selection pressures. The classical approach involves the analysis of nonsynonymous to synonymous replacements ratios (omega) for the branches of the seminal RNase evolutionary tree. The novel approach coupled these analyses with the mapping of substitutions on the folded structure of the protein. These analyses suggest that seminal RNase diverged during much of its history after divergence from pancreatic RNase as a functioning protein, followed by homoplastic inactivations to create pseudogenes in multiple ruminant lineages. Further, they are consistent with adaptive evolution only in the most recent episode leading to the gene in modern oxen. These conclusions contrast sharply with the view, cited widely in the literature, that seminal RNase decayed after its formation by gene duplication into an inactive pseudogene, whose lesions were repaired in a reactivation event. Further, the 2 approaches, omega estimation and mapping of replacements on the protein structure, were compared by examining their utility for establishing the functional status of the seminal RNase genes in 2 deer species. Hog and roe deer share common lesions, which strongly suggests that the gene was inactive in their last common ancestor. In this specific example, the crystallographic approach made the correct implication more strongly than the omega approach. Studies of this type should contribute to an integrated framework of tools to assign functional and nonfunctional episodes to recently created gene duplicates and to understand more broadly how gene duplication leads to the emergence of proteins with novel functions.

Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages
Yang, ZY; Sismour, AM; Benner, SA
Nucl. Acids Res.35(9) 3118-3127 (2007)
<Abstract>

The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re- examined in this work. The results presented here show that exonuclease III degrades single- stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned ( assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the ( R) stereoisomer of nucleoside alpha- thiotriphosphates [ and not the ( S) diastereomer], a conclusion inferred two decades ago by examination of five Family- A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III - thiotriphosphate - polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.

Enzymatic incorporation of a third nucleobase pair
Yang, ZY; Sismour, AM; Sheng, PP; Puskar, NL; Benner, SA
Nucl. Acids Res.35(13) 4238-4249 (2007)
<Abstract>

DNA polymerases are identified that copy a nonstandard nucleotide pair joined by a hydrogen bonding pattern different from the patterns joining the dA:T and dG:dC pairs. 6-Amino-5-nitro3-(l'-p-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) implements the non-standard 'small' donordonor-acceptor (pyDDA) hydrogen bonding pattern. 2-Amino-8-(1-beta-D-2'-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-4 (8H)-one [dP) implements the 'large' acceptor-acceptor-donor (puAAD) pattern. These nucleobases were designed to present electron density to the minor groove, density hypothesized to help determine specificity for polymerases. Consistent with this hypothesis, both dZTP and dPTP are accepted by many polymerases from both Families A and B. Further, the dZ:dP pair participates in PCR reactions catalyzed by Taq, Vent (exo(-)) and Deep Vent (exo-) polymerases, with 94.4%, 97.5% and 97.5%, respectively, retention per round. The dZ:dP pair appears to be lost principally via transition to a dC:dG pair. This is consistent with a mechanistic hypothesis that deprotonated dZ (presenting a pyDAA pattern) complements dG (presenting a puADD pattern), while protonated dC (presenting a pyDDA pattern) complements dP (presenting a puAAD pattern). This hypothesis, grounded in the Watson-Crick model for nucleobase pairing, was confirmed by studies of the pH-dependence of mismatching. The dZ:dP pair and these polymerases, should be useful in dynamic architectures for sequencing, molecular-, systems- and synthetic-biology.

The origin of proteins and nucleic acids
Ricardo, A; Benner, SA
Planets and Life: The Emerging Science of Astrobiology, ed. Woodruff T. Sullivan and John A. Baross, Cambridge University Press 154-173 (2007)

Alien biochemistries
Ward, PD; Benner, SA
Planets and Life: The Emerging Science of Astrobiology, ed. Woodruff T. Sullivan and John A. Baross, Cambridge University Press 537-544 (2007)

Inferred thermophily of the Last Universal Ancestor based on estimated amino acid composition
Brooks, DJ; Gaucher, EA
Ancestral Sequence Reconstruction, ed. David A. Liberles, Oxford University Press 200-207 (2007)
<Abstract>

The environmental temperature of the last universal ancestor (LUA) of all extant organisms is the subject of heated debate. Because the amino acid composition of proteins differs between mesophiles and thermophiles, the inferred amino acid composition of proteins in the LUA could be used to classify it as one or the other. We applied expectation maximization (EM) to estimate the amino acid composition of a set of thirty-one proteins in the LUA based on alignments of their modern day descendants, a phylogenetic tree relating those descendants and a model of evolution. Separate estimates of amino acid composition in LUA proteins were derived using modern day sequences of eight mesophilic species, eight thermophilic species or the sixteen species combined. We show that the relative mean Euclidean distance between the amino acid composition in one species and that of a set of mesophiles or thermophiles can be employed as a classifier with 100% accuracy. Applying this classifier to the estimated amino acid composition of the ancestral protein set in the LUA, we find it to be classified as a thermophile even when only the proteins of mesophilic species are used to derive the estimate. Based on the estimated amino acid composition of proteins in the LUA, we infer that it was a thermophile. We discuss our findings in the context of previous data pertaining to the OGT of the LUA, particularly the inferred G + C content of its rRNA. We conclude that the gathering evidence strongly supports a thermophilic LUA.

Crystallographic snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases
Berthold, CL; Toyota, CG; Moussatche, P; Wood, MD; Leeper, F; Richards, NGJ; Lindqvist, Y
Structure15(7) 853-861 (2007)
<Abstract>

Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycled and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate alpha-carbon for attack by the ThDP-C2 atom. The second structure presented shows a covalent reaction intermediate after decarboxylation, interpreted as being nonplanar. Finally, the structure of a product complex is presented. In accordance with mutagenesis data, no side chains of the enzyme are implied to directly participate in proton transfer except the glutamic acid (Glu-56), which promotes formation of the 1',4'-iminopyrimidine tautomer of ThDP needed for activation.

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development, cryotolerance and survival to term
Moore, K; Rodriguez-Sallaberry, CJ; Kramer, JM; Johnson, S; Wroclawska, E; Goicoa, S; Niasari-Nasalaji, A
Theriogenology68(9) 1316-1325 (2007)
<Abstract>

In this study, we evaluated a serum replacer (SR; Knockout SO (R), Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment I compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0) Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05) Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg +/- 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture. (C) 2007 Elsevier Inc. All rights reserved.

Molecular evolutionary models to guide experiments in protein engineering and directed evolution
Gaucher, EA
TBD (2007) In review

Montmorillonite protection of an UV-irradiated hairpin ribozyme. Evolution of the RNA world in a mineral environment
Biondi E., Branciamore S., Maurel M.-C., and Gallori E.
BMC Evol. Biol. (2007) 7(Suppl 2):S2

Catalytic activity of hammerhead ribozymes in a clay mineral environment: implications for the RNA World
Biondi E., Branciamore S., Fusi L., Gago, and Gallori E.
Gene (2007) 389:10-18

A major zebrafish polymorphism resource for genetic mapping
Bradley KM, Elmore JB, Breyer JP, Yaspan BL, Jessen JR, Knapik EW, Smith JR
Genome Biol, BioMed Central (2007) 8(4):R55
<Abstract>

We have identified 645,088 candidate polymorphisms in zebrafish and observe a single nucleotide polymorphism (SNP) validation rate of 71% to 86%, improving with polymorphism confidence score. Variant sites are non-random, with an excess of specific novel T- and A-rich motifs. We positioned half of the polymorphisms on zebrafish genetic and physical maps as a resource for positional cloning. We further demonstrate bulked segregant analysis using the anchored SNPs as a method for high-throughput genetic mapping in zebrafish.

Molecular Paleoscience: Systems Biology from the Past
Benner, SA; Sassi, SO; Gaucher, EA
Adv. Enzymol. Relat. Areas Mol. Biol.75 (2006)

Synthesis and antiviral activity of 7-deazaneplanocin A against orthopoxviruses (vaccinia and cowpox virus)
Arumugham, B; Kim, HJ; Prichard, MN; Kern, ER; Chu, CK
Bioorg. Med. Chem. Lett.16(2) 285-287 (2006)
<Abstract>

An efficient method for the synthesis of 7-deazaneplanocin A (2) has been accomplished by the condensation of cyclopentenol 3 with 6-chloro-7-deazapurine followed by subsequent functional group manipulations. The synthesized 7-deazaneplanocin A (2) exhibited potent antiviral activity against cowpox and vaccinia viruses without cytotoxicity in HFF cells. (c) 2005 Elsevier Ltd. All rights reserved.

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins
Bradley, ME; Benner, SA
BMC Bioinformatics7 89 (2006)
<Abstract>

Background: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Analysis of transitions at two-fold redundant sites in mammalian genomes. Transition redundant approach-to-equilibrium (TREx) distance metrics
Li, T; Chamberlin, SG; Caraco, MD; Liberles, DA; Gaucher, EA; Benner, SA
BMC Evol. Biol.6 25 (2006)
<Abstract>

Background: The exchange of nucleotides at synonymous sites in a gene encoding a protein is believed to have little impact on the fitness of a host organism. This should be especially true for synonymous transitions, where a pyrimidine nucleotide is replaced by another pyrimidine, or a purine is replaced by another purine. This suggests that transition redundant exchange ( TREx) processes at the third position of conserved two-fold codon systems might offer the best approximation for a neutral molecular clock, serving to examine, within coding regions, theories that require neutrality, determine whether transition rate constants differ within genes in a single lineage, and correlate dates of events recorded in genomes with dates in the geological and paleontological records. To date, TREx analysis of the yeast genome has recognized correlated duplications that established a new metabolic strategies in fungi, and supported analyses of functional change in aromatases in pigs. TREx dating has limitations, however. Multiple transitions at synonymous sites may cause equilibration and loss of information. Further, to be useful to correlate events in the genomic record, different genes within a genome must suffer transitions at similar rates. Results: A formalism to analyze divergence at two fold redundant codon systems is presented. This formalism exploits two-state approach-to-equilibrium kinetics from chemistry. This formalism captures, in a single equation, the possibility of multiple substitutions at individual sites, avoiding any need to "correct" for these. The formalism also connects specific rate constants for transitions to specific approximations in an underlying evolutionary model, including assumptions that transition rate constants are invariant at different sites, in different genes, in different lineages, and at different times. Therefore, the formalism supports analyses that evaluate these approximations. Transitions at synonymous sites within two-fold redundant coding systems were examined in the mouse, rat, and human genomes. The key metric (f(2)), the fraction of those sites that holds the same nucleotide, was measured for putative ortholog pairs. A transition redundant exchange ( TREx) distance was calculated from f(2) for these pairs. Pyrimidine-pyrimidine transitions at these sites occur approximately 14% faster than purine-purine transitions in various lineages. Transition rate constants were similar in different genes within the same lineages; within a set of orthologs, the f(2) distribution is only modest overdispersed. No correlation between disparity and overdispersion is observed. In rodents, evidence was found for greater conservation of TREx sites in genes on the X chromosome, accounting for a small part of the overdispersion, however. Conclusion: The TREx metric is useful to analyze the history of transition rate constants within these mammals over the past 100 million years. The TREx metric estimates the extent to which silent nucleotide substitutions accumulate in different genes, on different chromosomes, with different compositions, in different lineages, and at different times.

Application of DETECTER, an Evolutionary Genomic Tool to Analyze Genetic Variation, to the Cystic Fibrosis Gene Family
Gaucher, EA; DeKee, DW; Benner, SA
BMC Genomics7 44 (2006)
<Abstract>

Background: The medical community requires computational tools that distinguish genetic differences having phenotypic impact within the vast number of mutations that do not. Tools that do this will become increasingly important for those seeking to use human genome sequence data to predict disease, make prognoses, and customize therapy to individual patients.
Results: An approach, termed DETECTER, is proposed to identify sites in a protein sequence where amino acid replacements are likely to have a significant effect on phenotype, including causing genetic disease. This approach uses a model-dependent tool to estimate the normalized replacement rate at individual sites in a protein sequence, based on a history of those sites extracted from an evolutionary analysis of the corresponding protein family. This tool identifies sites that have higher-than-average, average, or lower- than-average rates of change in the lineage leading to the sequence in the population of interest. The rates are then combined with sequence data to determine the likelihoods that particular amino acids were present at individual sites in the evolutionary history of the gene family. These likelihoods are used to predict whether any specific amino acid replacements, if introduced at the site in a modern human population, would have a significant impact on fitness. The DETECTER tool is used to analyze the cystic fibrosis transmembrane conductance regulator (CFTR) gene family.
Conclusions: In this system, DETECTER retrodicts amino acid replacements associated with the cystic fibrosis disease with greater accuracy than alternative approaches. While this result validates this approach for this particular family of proteins only, the approach may be applicable to the analysis of polymorphisms generally, including SNPs in a human population.

The diverse biological functions of phosphatidylinositol transfer proteins in eukaryotes
Phillips, SE; Vincent, P; Rizzieri, KE; Schaaf, G; Bankaitis, VA; Gaucher, EA
Crit. Rev. Biochem. Mol. Biol.41(1) 21-49 (2006)
<Abstract>

Phosphatidylinositol/phosphatidylcholine transfer proteins (PITPs) remain largely functionally uncharacterized, despite the fact that they are highly conserved and are found in all eukaryotic cells thus far examined by biochemical or sequence analysis approaches. The available data indicate a role for PITPs in regulating specific interfaces between lipid-signaling and cellular function. In this regard, a role for PITPs in controlling specific membrane trafficking events is emerging as a common functional theme. However, the mechanisms by which PITPs regulate lipid-signaling and membrane-trafficking functions remain unresolved. Specific PITP dysfunctions are now linked to neurodegenerative and intestinal malabsorbtion diseases in mammals, to stress response and developmental regulation in higher plants, and to previously uncharacterized pathways for regulating membrane trafficking in yeast and higher eukaryotes, making it clear that PITPs are integral parts of a highly conserved signal transduction strategy in eukaryotes. Herein, we review recent progress in deciphering the biological functions of PITPs, and discuss some of the open questions that remain.

Reaction of tetramethylimidazol-2-ylidene with (Tp(tBu,Me))YbE(thf) (E=I, CH2SMe3): simple adduct and a hydrocarbyl tethered carbene ligand
Ferrence, GM; Arduengo, AJ; Jockisch, A; Kim, HJ; McDonald, R; Takats, J
J. Alloys Compd.418 184-188 (2006)
<Abstract>

Treatment of (Tp(tBu,Me))YbE(thf) (E=I (1), CH2SiMe3 (2)) with tetramethylimidazol-2-ylidene (ImMe(4)) resulted in very different outcomes depending on the nature of the anionic ligand E. ImMe(4) acts as a simple Lewis base toward 1 resulting in substitution of the thf (tetrahydrofuran) ligand and formation of (Tp(tBu,Me))YbI(ImMe(4)) (3). However, reaction with 2, in addition to displacement of thf, proceeded by metalation of one of the N-CH3 substituents of ImMe(4) and gave (Tp(tBu,Me))Yb(ImMe(4))(CH2N(C(CH3)C(CH3)N(CH3)C) (4), featuring a hydrocarbyl tethered carbene ligand. The X-ray structures of 3 and 4 are reported. (c) 2006 Published by Elsevier B.V.

Orthogonal activation of the reengineered A(3) adenosine receptor (neoceptor) using tailored nucleoside agonists
Gao, ZG; Duong, HT; Sonina, T; Kim, SK; Van Rompaey, P; Van Calenbergh, S; Mamedova, L; Kim, HO; Kim, MJ; Kim, AY; Liang, BT; Jeong, LS; Jacobson, KA
J. Med. Chem.49(9) 2689-2702 (2006)
<Abstract>

An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor ( neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A(3) adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3' : amino, aminomethyl, azido, guanidino, ureido; and at 5' : uronamido, azidodeoxy. N-6-Variations included 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N-6-3-iodobenzyl-3'-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC50 = 0.18 mu M) or phospholipase D in chick primary cardiomyocytes, both mediated by a mutant ( H272E), but not the wild-type, A(3)AR. The affinity enhancements for 10 and the corresponding 3'-acetamidomethyl analogue 6 were > 100-fold and > 20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A(3)AR (EC50 of 1.0 mu M), but had no effect on the H272E mutant A(3)AR (100 mu M). Compound 10 was inactive at human A(1), A(2A), and A(2B)ARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.

2-Hydroxymethylboronate as a Reagent To Detect Carbohydrates: Application to the Analysis of the Formose Reaction
Ricardo, A; Frye, F; Carrigan, MA; Tipton, JD; Powell, DH; Benner, SA
J. Org. Chem.71(25) 9503-9505 (2006)
<Abstract>

2-Hydroxymethylphenylboronate is described as a reagent that converts neutral 1,2-diols, as found in simple carbohydrates, into 1:1 anionic complexes that are easily detected by Fourier transform ion cyclotron resonance mass spectrometry. The value of this reagent was demonstrated through its application to analyze complex mixtures of carbohydrates formed in the formose process, often cited as a way that biologically significant carbohydrates might have been generated from formaldehyde under prebiotic conditions. Coupled with isotope studies, the reagent shows that the simplest autocatalytic cycle for the consumption of formaldehyde in this process cannot account for the bulk consumption of formaldehyde.

Zinc in yeast: mechanisms involved in homeostasis
Regalla, LM; Lyons, TJ
Molecular Biology of Metal Homeostasis and Detoxification: From Microbes to Man, ed. Springer, Markus J. Tamás and Enrico Martinoia 37-58 (2006)

Dynamic assembly of primers on nucleic acid templates
Leal, NA; Sukeda, M; Benner, SA
Nucl. Acids Res.34 4702-4710 (2006)
<Abstract>

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of 1 µM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern
Yang, ZY; Hutter, D; Sheng, PP; Sismour, AM; Benner, SA
Nucl. Acids Res.34(21) 6095-6101 (2006)
<Abstract>

To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin -4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA.

A review: Synthesis of aryl C-glycosides via the heck coupling reaction
Wellington, KW; Benner, SA
Nuc. Nuc. Nuc. acids25(12), Taylor & Francis Group 1309-1333 (2006)
<Abstract>

Looking for the primordial genetic honeycomb
Gallori E., Biondi E., and Branciamore S.
Orig. Life Evol. Biosph., Springer (2006) 36:493-499

Desorption/ionization on porous silicon mass spectrometry studies on pentose-borate complexes
Li, Q; Ricardo, A; Benner, SA; Winefordner, JD; Powell, DH
Anal. Chem.77(14) 4503-4508 (2005)
<Abstract>

Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) was used to investigate the binding affinities between aldopentose isomers and boron. Boron has been recognized for its importance in pentose synthesis and stabilization in prebiotic conditions. Boron may also account for the fact that ribose, among other aldopentoses, is the favored building block in RNA synthesis. This research started with the detection of aldopentoses in the positive mode through cationization and the aldopentose-borate complexes in the negative mode. Then two competition schemes, one using a pentose structure analogue and the other using C-13-labeled ribose, were designed to compare the relative binding affinities of four aldopentoses (xylose, lyxose, arabinose, and ribose) to boron. Both approaches determined the binding preference to be ribose > lyxose > arabinose > xylose. This work illustrates the potential of DIOS-MS in the analyses of nonvolatile, small molecules in delicate chemical equilibria. Without externally introduced matrices, background signals are not a limiting factor. Furthermore, the possible dramatic change of pH associated with the matrix introduction, which may disturb the equilibria of interest, is avoided.

Phylogenomic approaches to common problems encountered in the analysis of low copy repeats: The sulfotransferase IA gene family example
Bradley, ME; Benner, SA
BMC Evol. Biol.5 22 (2005)
<Abstract>

Background: Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs). Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT) IA genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage. Results: Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULTIA paralogs defined a new LCR family. The stem hominoid SULTIA progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULTIA expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project) has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript. Conclusion: SULTIA genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently) one after the divergence of chimpanzees and humans. Thus, LCRs may provide a means for amplifying genes (and other genetic elements) that are adaptively useful. Being located on and among LCRs, however, could make the human SULTIA genes susceptible to further duplications or deletions resulting in 'genomic diseases' for some individuals. Pharmacogenomic studies of SULTIAsingle nucleotide polymorphisms, therefore, should also consider examining SULTIA copy number variability when searching for genotype-phenotype associations. The latest duplication is, however, only a substantiated hypothesis; an alternative explanation, disfavored by the majority of evidence, is that the duplication is an artifact of incorrect genome assembly.

One-pot glycosylation (OPG) for the chemical synthesis of oligosaccharides
Yu, B; Yang, ZY; Cao, HZ
Curr. Org. Chem.9(2) 179-194 (2005)
<Abstract>

This review provides a comprehensive survey of the "one pot glycosylation" (OPG) strategy for the chemical synthesis of oligosaccharides, covering literatures from the first example reported by Kahne and Raghavan in 1993 through May 2003. The essence of the OPG is to distinguish the reactivity difference of a pair of the glycosylation donors or acceptors so as to carry out two glycosylation steps sequentially without purification of the first-step coupling product. Accordingly, the literature reports are grouped based on the major stereoelectronic factors causing the reactivity differences, those include the "armed-disarmed effect", "orthogonality of leaving groups", "distinguishable acceptors", and "the hybrid". "The hybrid" OPG procedure takes advantage of a combination of the reactivity disparity of a set of the armed-disarmed donors, orthogonal leaving groups, as well as acceptors so as to proceed three or more steps of glycosylation sequentially in one pot. Relevant conception and exploitation of the reactivity differences of the donors and acceptors in the synthesis of oligosaccharides, which finally evolve the OPG or advance parallelly, are briefly described at the beginning.

Synthetic biology
Sismour, AM; Benner, SA
Expert Opin. Biol. Ther.5(11) 1409-1414 (2005)
<Abstract>

Chemistry is a broadly powerful discipline in contemporary science because it has the ability to create new forms of the matter that it studies. By doing so, chemistry can test models that connect molecular structure to behaviour without having to rely on what nature has provided. This creation, known as synthesis', began to be applied to living systems in the 1980s as recombinant DNA technologies allowed biologists to deliberately change the molecular structure of the microbes that they studied, and automated chemical synthesis of DNA became widely available to support these activities. The impact of the information that has emerged has made biologists aware of a truism that has long been known in chemistry: synthesis drives discovery and understanding in ways that analysis cannot. Synthetic biology is now setting an ambitious goal: to recreate in artificial systems the emergent properties found in natural biology. By doing so, it is advancing our understanding of the molecular basis of genetics in ways that analysis alone cannot. More practically, it has yielded artificial genetic systems that improve the healthcare of some 400,000 Americans annually. Synthetic biology is now set to take the next step, to create artificial Darwinian systems by direct construction. Supported by the National Science Foundation as part of its Chemical Bonding program, this work cannot help but generate clarity in our understanding of how biological systems work.

RNA interference induced by siRNAs modified with 4 '-thioribonucleosides in cultured mammalian cells
Hoshika, S; Minakawa, N; Kamiya, H; Harashima, H; Matsuda, A
FEBS Lett.579(14) 3115-3118 (2005)
<Abstract>

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Solution H-1 NMR confirmation of folding in short o-phenylene ethynylene oligomers
Jones, TV; Slutsky, MM; Laos, R; de Greef, TFA; Tew, GN
J. Am. Chem. Soc.127(49) 17235-17240 (2005)
<Abstract>

Oligomers based on an o-phenylene ethynylene (oPE) backbone with polar substituents have been synthesized using Sonogashira methods. Folding of these extremely short oligomers was confirmed via 1D and 2D (NOESY) NMR methods. Utilizing electron-rich and electron-poor phenylene building blocks, variations of these oPE oligomers have been synthesized to determine the folded stability of pi-rich vs pi-poor vs pi-rich pi-poor systems. Slight variations in temperature offer a route, aside from solvent denaturation, to probe the stability of the folded structure. This is the first report of an NMR solution characterization of folding for a PE backbone without hydrogen bonds.

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate
Berthold, CL; Moussatche, P; Richards, NGJ; Lindqvist, Y
J. Biol. Chem.280(50), ASBMB 41645-41654 (2005)
<Abstract>

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemi-hedrally twinned crystal to 1.73 angstrom resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.

Planetary systems biology
Benner, SA; Ricardo, A
Mol. Cell17(4) 471-472 (2005)
<Abstract>

Combining paleogenetics, protein engineering, synthetic biology, and metabolic modeling, a planetary biology perspective is brought to bear on adaptive evolutionary events in ancient bacteria.

A call for likelihood phylogenetics even when the process of sequence evolution is heterogeneous
Gaucher, EA; Miyamoto, MM
Mol. Phylogenet. Evol.37(3) 928-931 (2005)
<Abstract>

All methods of phylogenetic inference make assumptions about the underlying evolutionary process of their characters and it is these assumptions that determine their relative successes and failures in the estimation of the true phylogeny for a group. This dependency of phylogenetic accuracy and robustness on evolutionary assumptions has been most extensively studied for the classic case of Felsenstein (1978) and its four-taxon phylogeny with two long, unrelated, terminal branches interspersed with two short ones. Given this model phylogeny, "long branch attraction" can occur and thereby lead to the convergence of a phylogenetic method onto an incorrect tree with the two long and two short terminal branches directly connected rather than interspersed. The extent to which a particular phylogenetic method is susceptible to this problem depends on what assumptions it makes about the evolution of the characters and data themselves.

The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system
Sismour, AM; Benner, SA
Nucl. Acids Res.33 5640-5646 (2005)
<Abstract>

Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC-isoG pair. The fidelity-per-round of amplification was found to be approximately 98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only approximately 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.

Synthesis of 3'-ureidoadenosines and their high binding affinity at the mutant A3 adenosine receptor
Kim, AY; Chung, HO; Kim, MJ; Chun, MW; Lee, KM; Jacobson, KA; Jeong, LS
Nucleic Acids Res. Suppl.49 105-106 (2005)

Resurrecting ancestral alcohol dehydrogenases from yeast
Thomson, JM; Gaucher, EA; Burgan, MF; De Kee, DW; Li, T; Aris, JP; Benner, SA
Nature Genet.37(6) 630-635 (2005)
<Abstract>

Modern yeast living in fleshy fruits rapidly convert sugars into bult ethanol through pyruvate. Pyruvate loses carbon dioxide to become acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. Because many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the reconstruction of the last common ancestor of Adh1 and Adh2, called AdhA. The kinetic behavior of AdhA suggests that it was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used AdhA to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

Synthetic Biology
Sismour, AM; Benner, SA
Nat. Rev. Genet.6 533-543 (2005)
<Abstract>

Synthetic biologists come in two broad classes. One uses unnatural molecules to reproduce emergent behaviours from natural biology, with the goal of creating artificial life. The other seeks interchangeable parts from natural biology to assemble into systems that function unnaturally. Either way, a synthetic goal forces scientists to cross uncharted ground to encounter and solve problems that are not easily encountered through analysis. This drives the emergence of new paradigms in ways that analysis cannot easily do. Synthetic biology has generated diagnostic tools that improve the care of patients with infectious diseases, as well as devices that oscillate, creep and play tic-tac-toe.

Synthesis of 3 '-ureidoadenosine analogues and their binding affinity to the A(3) adenosine receptor
Chun, MW; Lee, HW; Kim, AY; Kim, MJ; Kim, HO; Gao, ZG; Jacobson, KA; Jeong, LS
Nuc. Nuc. Nuc. acids24, Taylor & Francis Group 1119-1121 (2005)
<Abstract>

Novel 3'-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose in order to lead to stronger hydrogen bonding at the A(3) adenosine receptor than the corresponding 3'-aminoadenosine derivatives. However, all synthesized 3'-ureidoadenosine analogues have lost their binding affinities to the all subtypes of adenosine receptors, indicating that bulky 3'-urea moiety led to conformational distortion.

Synthesis of 3 '-deoxy-3 '-C-hydroxym ethyl analogues of tiazofurin and ribavirin
Chun, MW; Kim, MJ; Shin, JH; Jeong, LS
Nuc. Nuc. Nuc. acids24, Taylor & Francis Group 975-977 (2005)

Synthesis of homo-N-nucleoside with 1,2,4-triazole-3-carboxamide
Chun, MW; Kim, JH; Kim, MJ; Kim, BR; Jeong, LS
Nuc. Nuc. Nuc. acids24, Taylor & Francis Group 979-981 (2005)
<Abstract>

On the basis of Potent biological activities of ribavirin and homo-N-nucleosides, a novel homo-N-1,2,4-triazole-3-carboxamide derivative I was synthesized starting from 2,3,5-tri-O-benzoylribofuranosyl- I-acetate as a potential antiviral agent.

Synthesis and biological evaluation of novel isonucleosides with 1,2,4-triazole-3-carboxamide
Kim, MJ; Chung, SY; Chun, MW
Synth. Commun.35(20) 2653-2663 (2005)
<Abstract>

Novel 1,2,4-triazole isonucleosides (1 and 2) were efficiently synthesized starting from D-ribose and D-xylose, respectively. The key steps were condensation of cyclic sulfate 8 with methyl-1,2,4-triazole-3-carboxylate and nucleophilic displacement of the tosylate 15 with methyl-1,2,4-triazole-3-carboxylate, respectively.

Understanding nucleic acids using synthetic chemistry
Benner, SA
Acc. Chem. Res.37(10) 784-797 (2004)
<Abstract>

This Account describes work done in these laboratories that has used synthetic, physical organic, and biological chemistry to understand the roles played by the nucleobases, sugars, and phosphates of DNA in the molecular recognition processes central to genetics. The number of nucleobases has been increased from 4 to 12, generating an artificially expanded genetic information system. This system is used today in the clinic to monitor the levels of HIV and hepatitis C viruses in patients, helping to manage patient care. Work with uncharged phosphate replacements suggests that a repeating charge is a universal feature of genetic molecules operating in water and will be found in extraterrestrial life (if it is ever encountered). The use of ribose may reflect prebiotic processes in the presence of borate-containing minerals, which stabilize ribose formed from simple organic precursors. A new field, synthetic biology, is emerging on the basis of these experiments, where chemistry mimics biological processes as complicated as Darwinian evolution.

Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways
West, CM; van der Wel, H; Sassi, S; Gaucher, EA
Biochim. Biophys. Acta1673 29-44 (2004)
<Abstract>

The Skp1 protein, best known as a subunit of E3(SCF)-ubiquitin ligases, is subject to complex glycosylation in the cytoplasm of the cellular slime mold Dictyostelium. Pro143 of this protein is sequentially modified by a prolyl hydroxylase and five soluble glycosyltransferases (GT), to yield the structure Galalpha1,Galalpha1,3Fucalpha1,2Galbeta1,3GlcNAcalpha1-HyPro143. These enzymes are unusual in that they are expressed in the cytoplasmic compartment of the cell, rather than the secretory pathway where complex glycosylation of proteins usually occurs. The first enzyme in the pathway appears to be related to the soluble animal prolyl 4-hydroxylases (P4H), which modify the transcriptional factor subunit HIF-1alpha in the cytoplasm, and more distantly to the P4Hs that modify collagen and other proteins in the rER, based on biochemical and informatics analyses. The soluble alphaGlcNAc-transferase acting on Skp1 has been cloned and is distantly related to the mucin-type polypeptide N-acetyl-alpha-galactosaminyltransferase in the Golgi of animals. Its characterization has led to the discovery of a family of related polypeptide N-acetyl-alpha-glucosaminyltransferases in the Golgi of selected lower eukaryotes. The Skp1 GlcNAc is extended by a bifunctional diglycosyltransferase that sequentially and apparently processively adds beta1,3Gal and alpha1,2Fuc. Though this structure is also formed in the animal secretory pathway, the GTs involved are dissimilar. Conceptual translation of available genomes suggests the existence of this kind of complex cytoplasmic glycosylation in other eukaryotic microorganisms, including diatoms, oomycetes, and possibly Chlamydomonas and Toxoplasma, and an evolutionary precursor of this pathway may also occur in prokaryotes. (C) 2004 Elsevier B.V. All rights reserved.

Quantitative analysis of a RNA-cleaving DNA catalyst obtained via in vitro selection
Carrigan, MA; Ricardo, A; Ang, DN; Benner, SA
Biochemistry43(36), ACS 11446-11459 (2004)
<Abstract>

In vitro selections performed in the presence of Mg2+ generated DNA sequences capable of cleaving an internal ribonucleoside linkage. Several of these, surprisingly, displayed intermolecular catalysis and catalysis independent of Mg2+, features that the selection protocol was not explicitly designed to select. A detailed physical organic analysis was applied to one of these DNAzymes, termed 614. First, the progress curve for the reaction was dissected to identify factors that prevented the molecule from displaying clean first-order transformation kinetics and 100% conversion. Several factors were identified and quantitated, including (a) competitive intra- and intermolecular rate processes, (b) alternative reactive and unreactive conformations, and (c) mutations within the catalyst. Other factors were excluded, including "approach to equilibrium" kinetics and product inhibition. The possibility of complementary strand inhibition was demonstrated but was shown to not be a factor under the conditions of these experiments. The rates of the intra- and intermolecular processes were compared, and saturation models for the intermolecular process were built. The rate-limiting step for the intermolecular reaction was found to be the association/ folding of the enzyme with the substrate and not the cleavage step. The DNAzyme 614 is more active in trans than in cis and more active at temperatures below the selection temperature than at the selection temperature. Many of these properties have not been reported in similar systems; these results therefore expand the phenomenology known for this class of DNA-based catalysts. A brief survey of other catalysts arising from this selection found other Mg2+-independent DNAzymes and provided a preliminary view of the ruggedness of the landscape, relating function to structure in sequence space. Hypotheses are suggested to account for the fact that a selection in the presence of Mg2+ did not exploit this Mg2+. This study of a specific catalytically active DNAzyme is an example of studies that will be necessary generally to permit in vitro selection to help us understand the distribution of function in sequence space.

Oligodeoxynucleotides having a loop consisting of 3 '-deoxy-4 '-C-(2-hydroxyethyl)thymidines form stable hairpins
Yamamoto, Y; Shuto, S; Tamura, Y; Kodama, T; Hoshika, S; Ichikawa, S; Ueno, Y; Ohtsuka, E; Komatsu, Y; Matsuda, A
Biochemistry43(27), ACS 8690-8699 (2004)
<Abstract>

Components that form stable hairpin loops are highly useful for the development of functional DNA and RNA molecules. We have designed and synthesized a sugar-modified thymidine analogue, 3'-deoxy-4'-C-(2-hydroxyethyl)thymidine (X), as a nucleosidic loop component stabilizing the hairpin structure. The ODNs I-1-4, 5'-d[CGAACG-X-n-CGTTCG]-3' (I-1, n = 1; I-2, n = 2; I-3, n = 3; I-4, n = 4), forming the hairpin loop structures, of which the loop moiety consisted of the analogue X, and also the corresponding unmodified ODNs II-1-4, 5'-d[CGAACG-T-n-CGTTCG]-3' (II-1, n = 1; II-2, n = 2; II-3, n = 3; II-4, n = 4), having a thymidine loop, were synthesized by the phosphoramidite method. The melting temperatures (T m) of the ODNs I-1-4 containing X in the loop moiety at 5 muM were 67.1, 68.1, 73.0, and 69.3 degreesC, respectively, and those of the control natural ODNs II-1-4 were 65.3, 67.0, 69.2, and 68.8 degreesC, respectively. Thus, the ODNs I-1-4 formed a more thermally stable hairpin than the corresponding unmodified ODNs II-1-4 having an equal number of loop residues. The hairpin structures of the modified ODNs I-1-4 and the unmodified ODNs II-1-4 were investigated by CD spectroscopy and molecular mechanics calculations. These results showed that the 4'-branched nucleoside X can stabilize hairpin structures when it is present in the loop moiety, probably due to the flexibility of the one-carbon-elongated 4'-branched structure.

A novel method for estimating ancestral amino acid composition and its application to proteins of the Last Universal Ancestor.
Brooks, DJ; Fresco, JR; Singh, M
Bioinformatics20 2251-2257 (2004)
<Abstract>

Motivation: Knowledge of how proteomic amino acid composition has changed over time is important for constructing realistic models of protein evolution and increasing our understanding of molecular evolutionary history. The proteomic amino acid composition of the Last Universal Ancestor (LUA) of life is of particular interest, since that might provide insight into the early evolution of proteins and the nature of the LUA itself.
Results: We introduce a method to estimate ancestral amino acid composition that is based on expectation.maximization. On simulated data, the approach was found to be very effective in estimating ancestral amino acid composition, with accuracy improving as the number of residues in the dataset was increased. The method was then used to infer the amino acid composition of a set of proteins in the LUA. In general, as compared with the modern protein set, LUA proteins were found to be richer in amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those believed to have been unavailable or scarce. Additionally, we found the inferred amino acid composition of this protein set in the LUA to be more similar to the observed composition of the same set in extant thermophilic species than in extant mesophilic species, supporting the idea that the LUA lived in a thermophilic environment.
Availability: The program is available at http://compbio.cs.princeton.edu/ancestralaa

Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3 '-3 '-linked oligodeoxynucleotides composed of asymmetrical sequences
Hoshika, S; Ueno, Y; Kamiya, H; Matsuda, A
Bioorg. Med. Chem. Lett.14(12) 3333-3336 (2004)
<Abstract>

In this paper, we describe the synthesis of the 3'-3'-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3'-3'-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine-oligopurine sequences. (C) 2004 Elsevier Ltd. All rights reserved.

Design and synthesis of 3 '-ureidoadenosine-5 '-uronamides: effects of the 3 '-ureido group on binding to the A(3) adenosine receptor
Jeong, LS; Kim, MJ; Kim, HO; Gao, ZG; Kim, SK; Jacobson, KA; Chun, MW
Bioorg. Med. Chem. Lett.14(19) 4851-4854 (2004)
<Abstract>

On the basis of high binding affinity at the A(3) adenosine receptor of 3'-aminoadenosine derivatives with hydrogen bonding donor ability, novel 3'-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose in order to lead to stronger hydrogen bonding than the corresponding 3'-aminoadeno sine derivatives. However, the synthesized 3'-ureidoadenosine analogues were totally devoid of binding affinity, because 3'-urea moiety caused steric and electrostatic repulsions at the binding site of the A(3) adenosine receptor, leading to conformational distortion. (C) 2004 Elsevier Ltd. All rights reserved.

The planetary biology of cytochrome P450 aromatases
Gaucher, EA; Graddy, LG; Li, T; Simmen, RC; Simmen, FA; Schreiber, DR; Liberles, DA; Janis, CM; Benner, SA
BMC Biology2(1) 19 (2004)
<Abstract>

BACKGROUND: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.
RESULTS: Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases-enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene.
CONCLUSIONS: This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems.

Multiplexed genetic analysis using an expanded genetic alphabet
Johnson, SC; Marshall, DJ; Harms, G; Miller, CM; Sherrill, CB; Beaty, EL; Lederer, SA; Roesch, EB; Madsen, G; Hoffman, GL; Laessig, RH; Kopish, GJ; Baker, MW; Benner, SA; Farrell, PM; Prudent, JR
Clin. Chem.50(11) 2019-2027 (2004)
<Abstract>

Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. Methods: MultiCode(R) PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in similar to3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. Results: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. Conclusions: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting. (C) 2004 American Association for Clinical Chemistry.

Is there a common chemical model for life in the universe?
Benner, SA; Ricardo, A; Carrigan, MA
Curr. Op. Chem Biol.8(6) 672-689 (2004)
<Abstract>

A review of organic chemistry suggests that life, a chemical system capable of Darwinian evolution, may exist in a wide range of environments. These include non-aqueous solvent systems at low temperatures, or even supercritical dihydrogen-helium mixtures. The only absolute requirements may be a thermodynamic disequilibrium and temperatures consistent with chemical bonding. A solvent system, availability of elements such as carbon, hydrogen, oxygen and nitrogen, certain thermodynamic features of metabolic pathways, and the opportunity for isolation, may also define habitable environments. If we constrain life to water, more specific criteria can be proposed, including soluble metabolites, genetic materials with repeating charges, and a well defined temperature range.

Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase
Shaw, RW; Feller, JA; Bloom, LB
DNA Repair3(10) 1273-1283 (2004)
<Abstract>

Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme-DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision. (C) 2004 Elsevier B.V. All rights reserved.

Significance of cytoplasmic prolyl hydroxylation and complex glycosylation in the cellular slime mold Dictyostelium
West, CM; van der Wel, H; Sassi, S; Gaucher, E; Ercan, A
Glycobiology14(11) 1063-1063 (2004)

Expanding the genetic alphabet: Pyrazine nucleosides that support a donor-donor-acceptor hydrogen-bonding pattern
von Krosigk, U; Benner, SA
Helv. Chim. Acta87(6) 1299-1324 (2004)
<Abstract>

The 6-aminopyrazin-2(1H)-one, when incorporated as a pyrimidine-base analog into an oligonucleotide chain, presents a H-bond donor- donor- acceptor pattern to a complementary DNA or RNA strand. When paired with the corresponding acceptor-acceptor-donor purine in oligonucleotides, the heterocycle selectively contributes to the stability of the duplex, presumably by forming a base pair of Watson-Crick geometry joined by a nonstandard H-bonding pattern, expanding the genetic alphabet. Reported here is a short, high yielding, beta-D-selective synthesis of a 6-aminopyrazin-2(l H) -one nucleoside via the glycine riboside derivative 28. The key steps include a Wittig-Horner reaction of an appropriately protected ribose derivative (Scheme 10, 19 --> 21) followed by a Michael-like ring closure (Scheme 12, 30 --> la and 32 --> 1b). Thus, a variety of pyrazine nucleosides (Scheme 73) including the target 6-aminopyrazin-2(1H)-one riboside la, and its 5-methyl derivative 1b, 6-amino-5-methylpyrazin-2(1H)-one riboside, are obtained.

Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase
Leal, NA; Olteanu, H; Banerjee, R; Bobik, TA
J. Biol. Chem.279(46), ASBMB 47536-47542 (2004)
<Abstract>

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.

Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family
Moussatche, P; Klee, HJ
J. Biol. Chem.279(47), ASBMB 48734-48741 (2004)
<Abstract>

Receptors for the gaseous phytohormone ethylene show sequence similarity to bacterial two-component histidine kinases. These receptors are encoded by a multigene family that can be divided into subfamilies 1 and 2. It has been previously shown that a subfamily 1 Arabidopsis thaliana ethylene receptor, ETR1, autophosphorylates in vitro on a conserved histidine residue (1). However, sequence comparisons between the five ethylene receptor family members suggest that subfamily 2 members do not have all the motifs necessary for histidine kinase activity. Further, a tobacco subfamily 2 receptor, NTHK1, autophosphorylates on serines and threonines in vitro (2). Here we show that all five Arabidopsis ethylene receptor proteins autophosphorylate in vitro. We analyzed the nature of the phosphorylated amino acids by acid/base stability and bi-dimensional thin layer electrophoresis and demonstrated that unlike ETR1 all other ethylene receptors autophosphorylate predominantly on serine residues. ERS1, the only other subfamily 1 receptor, is able to phosphorylate on both histidine and serine residues in the presence of Mn2+. However, histidine autophosphorylation is lost when ERS1 is assayed in the presence of both Mg2+ and Mn2+, suggesting that this activity may not occur in vivo. Furthermore, mutation of the histidine residue conserved in two-component systems does not abolish serine autophosphorylation, eliminating the possibility of a histidine to serine phosphotransfer. Our biochemical observations complement the recently published genetic data that histidine kinase activity is not necessary for ethylene receptor function in plants and suggest that ethylene signal transduction does not occur through a phosphorelay mechanism.

Zinc and the Msc2 zinc transporter protein are required for endoplasmic reticulum function
Ellis, CD; Wang, FD; MacDiarmid, CW; Clark, S; Lyons, T; Eide, DJ
J. Cell. Biol.166(3) 325-335 (2004)
<Abstract>

In this report, we show that zinc is required for endoplasmic reticulum function in Saccharomyces cerevisiae. Zinc deficiency in this yeast induces the unfolded protein response (UPR), a system normally activated by unfolded ER proteins. Msc2, a member of the cation diffusion facilitator (CDF) family of metal ion transporters, was previously implicated in zinc homeostasis. Our results indicate that Msc2 is one route of zinc entry into the ER. Msc2 localizes to the ER when expressed at normal levels. UPR induction in low zinc is exacerbated in an msc2 mutant. Genetic and biochemical evidence indicates that this UPR induction is due to genuine ER dysfunction. Notably, we found that ER-associated protein degradation is defective in zinc-limited msc2 mutants. We also show that the vacuolar CDF proteins Zrc1 and Cot1 are other pathways of ER zinc acquisition. Finally, zinc deficiency up-regulates the mammalian ER stress response indicating a conserved requirement for zinc in ER function among eukaryotes.

Empirical analysis of protein insertions and deletions determining parameters for the correct placement of gaps in protein sequence alignments
Chang, MSS; Benner, SA
J. Mol. Biol.341(2) 617-631 (2004)
<Abstract>

To understand how protein segments are inserted and deleted during divergent evolution, a set of pairwise alignments contained exactly one gap, and therefore arising from the first insertion-deletion (indel) event in the time separating the homologs, was examined. The alignments showed that "structure breaking" amino acids (PGDNS) were preferred within and flanking gapped regions, as are two residues with hydrophilic side-chains (QE) that frequently occur at the surface of protein folds. Conversely, hydrophobic residues (FMILYVW) occur infrequently within and flanking the gapped region. These preferences are modestly different in protein pairs separated by an episode of adaptive evolution, than in pairs diverging under strong functional constraints. Surprisingly, regions near an indel have not evolved more rapidly than the sequence pair overall, showing no evidence that an indel event must be compensated by local amino acid replacement. The gap-lengths are best approximated by a Zipfian distribution, with the probability of a gap of length L decreasing as a function of L-1.8. These features are largely independent of the length of the gap and the extent of divergence (measured by both silent and non-silent sequence changes) separating the two proteins. Surprisingly, amino acid repeats were discovered in more than a third of the polypeptide segments in and around the gap. These correspond to repeats in the DNA sequence. This suggests that a signature of the mechanism by which indels occur in the DNA sequence remains in the encoded protein sequences. These data suggest specific tools to score gap placement in an alignment. They also suggest tools that distinguish true indels from gaps created by mistaken gene finding, including under-predicted and overpredicted introns. By providing mechanisms to identify errors, the tools will enhance the value of genome sequence databases in support of integrated paleogenomics strategies used to extract functional information in a post-genomic environment.

Artificial genetic systems: Exploiting the "aromaticity" formalism to improve the tautomeric ratio for isoguanosine derivatives
Martinot, TA; Benner, SA
J. Org. Chem.69(11) 3972-3975 (2004)
<Abstract>

The tautomerism of 2'-deoxy-7-deaza-isoguanosine (2) was studied and compared to that of 2'-deoxyisoguanosine (1). The fixed N-1-methyl (8) and O-methyl (4) derivatives were synthesized to represent the pure extremes of each tautomer. The replacement of the imidazole ring in 1 with a pyrrole ring in 2 makes the keto form in the latter more favored by 2 orders of magnitude (K-TAUT for 2 approximate to 10(3), as opposed to K-TAUT for 1 approximate to 10).

The Sup35 domains required for maintenance of weak, strong or undifferentiated yeast [PSI+] prions
Bradley, ME; Liebman, SW
Mol. Microbiol.51(6) 1649-1659 (2004)
<Abstract>

The Sup35 protein can exist in a non-infectious form or in various infectious forms called [PSI+] prion variants (or prion strains). Each of the different [PSI+] prion variants converts non-infectious Sup35 molecules into that prion variant's infectious form. One definition of a 'prion domain' is the minimal fragment of a prion protein that is necessary and sufficient to maintain the prion form. We now demonstrate that the Sup35 N region (residues 1-123), which is frequently referred to as the 'prion domain', is insufficient to maintain the weak or strong [PSI (+)] variants per se, but appears to maintain them in an 'undifferentiated'[PSI+] state that can differentiate into weak or strong [PSI+] variants when transferred to the full-length Sup35 protein. In contrast, Sup35 residues 1-137 are necessary and sufficient to faithfully maintain weak or strong [PSI+] variants. This implicates Sup35 residues 124-137 in the variant-specific maintenance of the weak or strong [PSI+] forms. Structure predictions indicate that the residues in the 124-137 region form an alpha-helix and that the 1-123 region may have beta structure. In view of these findings, we discuss a plausible molecular basis for the [PSI+] prion variants as well as the inherent difficulties in defining a 'prion domain'.

Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2 '-deoxyadenosine
Hendrickson, CL; Devine, KG; Benner, SA
Nucl. Acids Res.32(7) 2241-2250 (2004)
<Abstract>

Standard nucleobases all present electron density as an unshared pair of electrons to the minor groove of the double helix. Many heterocycles supporting artificial genetic systems lack this electron pair. To determine how different DNA polymerases use the pair as a substrate specificity determinant, three Family A polymerases, three Family B polymerases and three reverse transcriptases were examined for their ability to handle 3-deaza-2'-deoxyadenosine (c(3)dA), an analog of 2'-deoxyadenosine lacking the minor groove electron pair. Different polymerases differed widely in their interaction with c(3)dA. Most notably, Family A and Family B polymerases differed in their use of this interaction to exploit their exonuclease activities. Significant differences were also found within polymerase families. This plasticity in polymerase behavior is encouraging to those wishing to develop a synthetic biology based on artificial genetic systems. The differences also suggest either that Family A and Family B polymerases do not share a common ancestor, that minor groove contact was not used by that ancestor functionally or that this contact was not sufficiently critical to fitness to have been conserved as the polymerase families diverged. Each interpretation is significant for understanding the planetary biology of polymerases.

A novel replicating circular DNAzyme
Chen, F; Wang, RJ; Li, Z; Liu, B; Wang, XP; Sun, YH; Hao, DY; Zhang, J
Nucl. Acids Res.32(8) 2336-2341 (2004)
<Abstract>

10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10-23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz(7) and C-Dz(482), targeting the beta-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k(cat)/K-M) of 7.82 x 10(6) and 1.36 x 10(7) M-1.min(-1), respectively. Their dependence on divalent metal ions is similar to that found with linear 10-23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting beta-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.

Synthesis and physical and physiological properties of 4 '-thioRNA: application to post-modification of RNA aptamer toward NF-kappa B
Hoshika, S; Minakawa, N; Matsuda, A
Nucl. Acids Res.32(13) 3815-3825 (2004)
<Abstract>

We report herein full details of the preparation of 4'-thiouridine, -cytidine, -adenosine and -guanosine phosphoramidites based on our synthetic protocol via the Pummerer reaction. Fully modified 4'-thioRNAs containing four kinds of 4'-thioribonucleoside units were prepared according to the standard RNA synthesis. The T,, values and thermodynamic parameters of a series of duplexes were determined by UV melting and differential scanning calorimetry (DSC) measurements. The resulting overall order of thermal stabilities for the duplexes was 4'-thioRNA:4'-thioRNA >> 4'-thioRNA:RNA > RNA:RNA > RNA:DNA > 4'-thioRNA:DNA. In addition, it was shown that the dominant factor in the stability of the duplexes consisting of 4'-thioRNA was enthalpic in character. The CD spectra of not only 4'-thioRNA:RNA and 4'-thioRNA:4'-thioRNA but also 4'-thioRNA:DNA were all similar to those of duplexes in the A-conformation. The stability of 4'-thioRNA in human serum was 600 times greater than that of natural RNA. Neither the RNA:RNA nor the 4'-thioRNA: 4'-thioRNA duplexes were digested under the same conditions. The first example of a post-modification of an RNA aptamer by 4'-thioribonucleoside units was demonstrated. Full modification of the aptamer thioRNA3 resulted in complete loss of binding activity. In contrast, modifications at positions other than the binding site were tolerated without loss of binding activity. The post-modified RNA aptamer thioRNA5 was thermally stabilized and resistant toward nuclease digestion. The results presented in this paper will, it is hoped, contribute to the development of 4'-thioRNA as a new generation of artificial RNA.

PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1
Sismour, AM; Lutz, S; Park, JH; Lutz, MJ; Boyer, PL; Hughes, SH; Benner, SA
Nucl. Acids Res.32 728-735 (2004)
<Abstract>

As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond 'donor-acceptor-donor' pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond 'acceptor-donor-acceptor' pattern. This base pair fits the Watson-Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification.

Synthesis and biological evaluation of novel apio nucleosides with thiazole-4-carboxamide and 1,2,4-triazole-3-carboxamide
Kim, MJ; Jeong, LS; Kim, JH; Shin, JH; Chung, SY; Lee, SK; Chun, MW
Nuc. Nuc. Nuc. acids23(4), Taylor & Francis Group 715-724 (2004)
<Abstract>

In view of biological activities of azole nucleosides and apio-dideoxynucleoside, novel apio nucleoside analogues (1 and 2) with thiazole and triazole base moiety were synthesized using 2,3-O-isopropylidene-apio-beta-D-furanose (3), which was prepared from D-mannose.

Inhibition of ampicillin-resistant bacteria by novel mono-DNAzymes and di-DNAzyme targeted to beta-lactamase mRNA
Chen, F; Li, Z; Wang, RJ; Liu, B; Zeng, Z; Zhang, HY; Zhang, J
Oligonucleotides14(2) 80-89 (2004)
<Abstract>

In view of the weakness of antibiotics and the properties of antisense drugs, we applied DNAzymes to the field of drug resistance in bacteria. Two 10-23 mono-DNAzymes (Dz(1), Dz(2)) and a di-DNAzyme (Dz(1-2)) targeted to beta-lactamase mRNA were designed to determine to what degree the growth of ampicillin-resistant bacteria (TEM-1, TEM-3) was inhibited. All three DNAzymes can play a role both in vitro and in vivo. In vitro, they exhibited high catalytic efficiency (k(cat)/K-M) of 63.5, 91.1, and 30.8 pM(-1) (.) min(-1), respectively, under multiple-turnover conditions. In vivo, after 9 hours' incubation, the degree of inhibition of Dz(1), Dz(2), and Dz(1-2) for TEM-1 bacteria was 27.2%, 39.6%, and 57.7%, respectively, and that for TEM-3 bacteria was 39.1%, 44%, and 62.6%, respectively. Dz(1-2) showed the greatest inhibiting effect, demonstrating in vivo activity may be increased by constructing multiple-target DNAzymes. The results indicated a potential possibility for DNAzymes to act as a new type of antibacterial or a tool of gene functional analysis for prokaryocytes.

2 '-deoxycytidines carrying amino and thiol functionality: Synthesis and incorporation by vent (exo(-)) polymerase
Roychowdhury, A; Illangkoon, H; Hendrickson, CL; Benner, SA
Org. Lett.6(4) 489-492 (2004)
<Abstract>

The synthesis of 2'-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a butynyl linker is described. The corresponding triphosphates were then synthesized from the nucleoside and incorporated into oligonucleotides by Vent (exo(-)) DNA polymerase. The ability of Vent (exo(-)) polymerase to amplify oligonucleotides containing these functionalized cytidine derivatives in a polymerase chain reaction (PCR) was demonstrated for the amino-functionalized derivative.

Metalloregulation of yeast membrane steroid receptor homologs
Lyons, TJ; Villa, NY; Regalla, LM; Kupchak, BR; Vagstad, A; Eide, DJ
Proc. Natl. Acad. Sci. USA101(15) 5506-5511 (2004)
<Abstract>

Zinc is an essential micronutrient that can also be toxic. An intricate mechanism exists in yeast that maintains cellular zinc within an optimal range. The centerpiece of this mechanism is the Zap1p protein, a transcription factor that senses zinc deficiency and responds by up-regulating genes involved in zinc metabolism. A microarray screen for novel Zap1p target genes suggested a role in zinc homeostasis for four homologous yeast genes. The expression of two of these genes, YDR492w and YOL002c, suggested direct regulation by Zap1p, whereas the expression of YOL002c and a third homologous gene, YOL101c, was induced by high zinc. YDR492w and YOL002c are confirmed to be direct Zap1p target genes. The induction of YOL002c and YOL101c by toxic metal ion exposure is shown to be mediated by the Mga2p hypoxia sensor. Furthermore, YOL101c is induced by deletion of the Aft1p iron-responsive transcription factor. These three genes, along with a fourth yeast homolog, YLR023c, have phenotypic effects on zinc tolerance and Zap1p activity. Because of their metalloregulation, zinc-related phenotypes, and highly conserved motifs containing potential metal-binding residues, this family has been renamed the IZH gene family (implicated in Zinc Homeostasis). Furthermore, these genes are regulated by exogenous fatty acids, suggesting a dual role in lipid metabolism. The IZH genes encode membrane proteins that belong to a ubiquitous protein family that includes hemolysin III and vertebrate membrane steroid receptors. We propose that the IZH genes affect zinc homeostasis either directly or indirectly by altering sterol metabolism.

Borate minerals stabilize ribose
Ricardo, A; Carrigan, MA; Olcott, AN; Benner, SA
Science303(5655) 196-196 (2004)

Redesigning genetics
Benner, SA
Science306(5296) 625-626 (2004)

Mineral Surfaces as a Cradle of Primordial Genetic Material
Gallori E, Biondi E, Franchi M
Life in the Universe, ed. Seckbach J, Chela-Flores J, Owen T, Raulin F, Netherlands: Springer 145-148 (2004)
<Abstract>

Molecules which store genetic information (DNA and RNA) are central to all life on Earth. The formation of these complex macromolecules, and ultimately life, required specific conditions, including the synthesis and polymerization of precursors (nucleotides), the protection and persistence of information polymers in a changing environment, and the expression of the "biological potential" of the molecules, i.e. their capacity to multiply and evolve. Determining how these steps occurred and how the earliest genetic molecules originated on Earth is a problem that is far from being resolved. Recent observations on the synthesis of polynucleotides on clay surfaces, the resistance of clay-adsorbed nucleic acid molecules to environmental degradation and the biological activity of clay-adsorbed DNA and RNA molecules suggest that mineral surfaces could have played a crucial role in the prebiotic formation of the biomolecules basic to life.

Interpretive proteomics - finding biological meaning in genome and proteome databases
Benner, SA
Adv. Enz. Reg.43(12) 271-359 (2003)

PduP is a Coenzyme-A-Acylating Propionaldehyde Dehydrogenase Associated with the Polyhedral Bodies Involved in B12-dependent 1,2-propanediol Degradation by Salmonella enterica serovar Typhimurium LT2
Leal, NA; Havemann, GD; Bobik, TA
Arch. Microbiol.180(5) 353-361 (2003)
<Abstract>

Salmonella enterica forms polyhedral bodies involved in coenzyme-B12-dependent 1,2-propanediol degradation. Prior studies showed that these bodies consist of a proteinaceous shell partly composed of the PduA protein, coenzyme-B12-dependent diol dehydratase, and additional unidentified proteins. In this report, we show that the PduP protein is a polyhedral-body-associated CoA-acylating aldehyde dehydrogenase important for 1,2-propanediol degradation by S. enterica. A PCR-based method was used to construct a precise nonpolar deletion of the gene pduP. The resulting pduP deletion strain grew poorly on 1,2-propanediol minimal medium and expressed 105-fold less propionaldehyde dehydrogenase activity (0.011 micromol min(-1) mg(-1)) than did wild-type S. enterica grown under similar conditions (1.15 micromol min(-1) mg(-1)). An Escherichia coli strain was constructed for high-level production of His8-PduP, which was purified by nickel-affinity chromatography and shown to have 15.2 micromol min(-1) mg(-1) propionaldehyde dehydrogenase activity. Analysis of assay mixtures by reverse-phase HPLC and mass spectrometry established that propionyl-CoA was the product of the PduP reaction. For subcellular localization, purified His8-PduP was used as antigen for the preparation of polyclonal antiserum. The antiserum obtained was shown to have high specificity for the PduP protein and was used in immunogold electron microscopy studies, which indicated that PduP was associated with the polyhedral bodies involved in 1,2-propanediol degradation. Further evidence for the localization of the PduP enzyme was obtained by showing that propionaldehyde dehydrogenase activity co-purified with the polyhedral bodies. The fact that both Ado-B12-dependent diol dehydratase and propionaldehyde dehydrogenase are associated with the polyhedral bodies is consistent with the proposal that these structures function to minimize propionaldehyde toxicity during the growth of S. enterica on 1,2-propanediol.

The NASA astrobiology roadmap
Marais, DJD; Allamandola, LJ; Benner, SA; Boss, AP; Deamer, D; Falkowski, PG; Farmer, JD; Hedges, SB; Jakosky, BM; Knoll, AH; Liskowsky, DR; Meadows, VS; Meyer, MA; Pilcher, CB; Nealson, KH; Spormann, AM; Trent, JD; Turner, WW; Woolf, NJ; Yorke, HW
Astrobiology3(2) 219-235 (2003)
<Abstract>

The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: How does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own solar system, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high-priority efforts for the next 3-5 years. These 18 objectives are being integrated with NASA strategic planning.

First PCR amplification of DNA containing a nonstandard base pair A.
Lutz, S; Park, JH; Benner, SA
Biochemistry42(28), ACS 8598-8598 (2003)

Nucleosides and nucleotides. 218. Alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides using a purine motif
Hoshika, S; Ueno, Y; Matsuda, A
Bioconjugate Chem.14(3) 607-613 (2003)
<Abstract>

In this paper, we describe the synthesis of the X-X-linked TFOs that can form the antiparallel triplexes with the duplex DNA target by reverse Hoogsteen hydrogen bonds. Stability of the alternate-strand triplexes between these TFOs and the target DNAs was investigated using the electrophoretic mobility shift assay (EMSA). It was found that the alternate-strand triplexes were significantly stabilized by linking the TFO fragments with the pentaerythritol linker. And, unlike the alternate-strand triplexes composed of the pyrimidine motif, the terminal ammonium ion of the aminobutyl-linker and the intercalator of the TFOs did not contribute to the stability of the alternate-strand triplex comprised of the purine motif. We also tested the ability of the X-X-linked TFOs to inhibit cleavage of the duplex DNA target 17 by the restriction enzyme EcoT14I and found that the 3'-3'-Iinked TFOs 12 and 13 inhibited the cleavage by the enzyme more effectively than the unlinked decamer S. Thus, the TFOs linked with pentaerythritol may be useful as the antigene oligonucleotide to the DNA targets, which have alternating oligopyrimidine-oligopurine sequences.

Synthesis and biological evaluation of thymine nucleosides fused with 3 ',4 '-tetrahydrofuran ring
Kim, MJ; Kim, HO; Kim, HD; Kim, JH; Jeong, LS; Chun, MW
Bioorg. Med. Chem. Lett.13(20) 3499-3501 (2003)
<Abstract>

The pyrimidine nucleosides fused with 3',4'-tetrahydrofuran ring were successfully synthesized, starting from 1,2;5,6-di-O-isopropylidene-D-glucose and assayed for antiviral activities against HIV-1, HIV-2, EMCV, Cox. B3 and VSV. Thymine analogue (5) and its corresponding 2'-deoxy analogue (6) exhibited high cytotoxicity instead of giving antiviral activities. (C) 2003 Elsevier Ltd. All rights reserved.

Greater GNN pattern bias in sequence elements encoding conserved residues of ancient proteins may be an indicator of amino acid composition of early proteins.
Brooks, DJ; Fresco, JR
Gene303 177-185 (2003)
<Abstract>

The possibility that RNY pattern bias in extant sequences is a remnant of more pronounced bias of this type in early ancestors was investigated. To this end, conserved residues (those residues for which the inferred ancestral and known descendant amino acids are identical) and non-conserved residues of ancient proteins dating to the Last Universal Ancestor were identified within six species: two archaea, two eubacteria and two eukaryotes. Bias within sequence elements encoding each subset of residues, conserved and non-conserved, was then determined. In all species, GNN bias is greater within conserved than non-conserved sequence elements, whereas ANN is not. This difference is statistically significant in all six species examined. Since the relative mutability of the GNN-encoded amino acids does not explain the greater bias in conserved sequences, it is concluded that early sequences probably possessed a strong GNN bias. It is suggested that this bias may be a consequence of the GNN codons being the first introduced into the genetic code. Although NNY bias is also greater within conserved sequence elements of the six species, that difference is statistically significant in only half of them. Therefore, the evidence for early NNY bias remains inconclusive. The findings of this study do not support the proposal of Diaz-Lazcoz et al. (J. Mol. Biol. 250 (1995) 123) that the codons of the TCN four-codon block were the first assigned to serine during the evolution of the genetic code.

Evolutionary, structural and biochemical evidence for a new interaction site of the leptin obesity protein
Gaucher, EA; Miyamoto, MM; Benner, SA
Genetics163(4) 1549-1553 (2003)
<Abstract>

The Leptin protein is central to the regulation of energy metabolism in mammals. By integrating evolutionary, structural, and biochemical information, a surface segment, outside of its known receptor contacts, is predicted as a second interaction site that may help to further define its roles in energy balance and its functional differences between humans and other mammals.

Destabilizing interactions among [PSI+] and [PIN+] yeast prion variants
Bradley, ME; Liebman, SW
Genetics165(4) 1675-1685 (2003)
<Abstract>

The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi(-)] or [pin(-)], respectively, or the multiple infectious amyloid-like forms called [PSI+] or [PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.

Initiation of mucin-type O-glycosylation in lower eukaryotes (O-alpha-GlcNAc-type) and higher eukaryotes (O-alpha-GalNAc-type) is homologous
West, CM; Wang, F; van der Wel, H; Gaucher, E; Sassi, S; Metcalf, T; Heise, N; Mendonca-Previato, L; Previato, JO
Glycobiology13(11) 875-876 (2003)

A direct synthesis of nucleoside analogs homologated at the 3 '- and 5 '-positions
Schmidt, J; Eschgfaller, B; Benner, SA
Helv. Chim. Acta86(9) 2937-2958 (2003)
<Abstract>

A new route is presented to prepare analogs of nucleosides homologated at the 3'- and 5'-positions. This route, applicable to both the D- and L-enantiomeric forms, is suitable for the preparation of monomeric bis-homonucleosides needed for the synthesis of oligonucleotide analogs. It begins with the known monobenzyl ether 3 of pent-2-yne-1,5-diol, which is reduced to alkenol 4. Sharpless asymmetric epoxidation of 4, followed by opening of the epoxide 5 with allylmagnesium bromide, gives a mixture of diols 6 and 7 Protection of the primary alcohol as a silyl ether followed by treatment with OsO4, NalO(4), and mild acid in MeOH, followed by reduction, yields (2R,3R) {{[(tert-butyl)diphenylsilyl]oxy}methyl}tetrahydro-2-(2-hydroxyethyl)-5- methoxyfuran (=methyl 3-{{[(tert-butyl)diphenylsilyl]oxy}methyl}-2 3,5-trideoxy-alpha/beta-D-erythro-hexafuranoside: 10) (Scheme 1). Protected nucleobases are added to this skeleton with the aid of trimethylsilyl triflate (Scheme 2). The o-toluoyl (2-MeC6H4CO) and p-anisoyl (4-MeOC6H4CO) groups were used to protect the exocyclic amino group of cytosine. The bis-homonucleoside analogs 11 and 14a are then converted to monothiol derivatives suitable for coupling (Schemes 3 and 4) to oligonucleotide analogs with bridging S-atoms. This synthesis replaces a much longer synthesis for analogous nucleoside analogs that begins with diacetoneglucose (= 1,2:5,6-di-O-isopropylideneglucose), with the stereogenic centers in the final products derived from the Sharpless asymmetric epoxidation. The new route is useful for large-scale synthesis of these building blocks for the synthesis of oligonucleotide analogs.

Synthesis and properties of oligodeoxynucleotide analogs with bis(methylene) sulfone bridges
Eschgfaller, B; Schmidt, JG; Konig, M; Benner, SA
Helv. Chim. Acta86(9) 2959-2997 (2003)
<Abstract>

A convergent, solution-phase synthesis was developed for the bis(methylene) sulfone-bridged oligodeoxynucleotide analogs (SNA) 5'-d(HOCH2-Tso(2)Tso(2)Tso(2)Cso(2)Tso(2)Tso(2)Tso(2)T-CH2SO3-)-3' (35b) and 5'-d(HOCH2-Tso(2)Tso(2)Tso(2)Tso(2)Tso(2)Tso(2)Tso(2)T-CH2SO3-)-3' (34c) (SO2 corresponds to CH2SO2CH2 instead of OP(=O)(O-)(O). In these, the phosphodiester linkages are replaced by non-ionic bis(methylene) sulfone linkers. The general strategy involved convergent coupling of 3',5'-bishomo-beta-D-deoxyribonucleotide analogs functionalized at the 6'-end (=CH2-C(5')) as bromides or mesylates and at the CH2-C(3') position as thiols. with the resulting thioether being oxidized to the corresponding sulfone. A single charge was introduced at the terminal CH2-C(3') position of the octamers to increase their solubility in water. During the synthesis, it became apparent that the key intermediates generated secondary structures through either folding or aggregation in a variety of solvents. This generated unusual reactivity and was unique for very similar structures. For example, although the dimeric thiol d(BzOCH(2)-Tso(2)C-CH2SH) (14b) was a well-behaved synthetic intermediate, the tetrameric thiol d(TrOCH2-Tso(2)Tso(2)Tso(2)(10)C-CH2SH) derived from the corresponding thioacetate was rapidly converted to a disulfide by very small amounts of oxidant (28 --> 29, Scheme 6). while the analogous tetrameric thiol d(BzOCH(2)-Tso(2)TsTso(2)T-CH2SH) (26), differing only by a single heterocycle, was oxidized much more slowly (Bz = PhCO, Tr = Ph3C, to = 2-MeC6H4CO (at N-4 of dc)). The sequence-dependent reactivity, well known in many classes of natural products (including polypeptides), is not prominent in natural oligonucleotides. These results are discussed in light of the proposal that the repeating negative charge in nucleic acids is key to their ability to serve as genetic molecules, in particular, their capability to support Darwinian evolution. The ability of 5'-d(HOCH2-Tso(2)Tso(2)Tso(2)Cso(2)Tso(2)Tso(2)Tso(2)T-CH2SO3-)-3' (35b) to bind as a third strand to duplex DNA was also examined. No triple-helix-forming propensity was detected in this molecule.

Identification of the Human and Bovine ATP:cob(I)alamin Adenosyltransferase cDNAs Based on Complementation of a Bacterial Mutant
Leal, NA; Park, SD; Kima, PE; Bobik, TA
J. Biol. Chem.278(11), ASBMB 9227-9234 (2003)
<Abstract>

In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.

Expanding the genetic alphabet: Non-epimerizing nucleoside with the pyDDA hydrogen-bonding pattern
Hutter, D; Benner, SA
J. Org. Chem.68(25) 9839-9842 (2003)
<Abstract>

6-Amino-3-(2'-deoxy-beta-D-ribofuranosyl)-5-nitro-1H-pyridin-2-one (4), a C-glycoside exhibiting the nonstandard pgammaDDA hydrogen-bonding pattern, was synthesized via Heck coupling. The nitro group greatly enhances the stability of the nucleoside toward acid-catalyzed epimerization without leading to significant deprotonation of the heterocycle at physiological pH. These results make nucleoside 4 a promising candidate for an expanded genetic alphabet.

Synthetic biology with artificially expanded genetic information systems. From personalized medicine to extraterrestrial life
Benner, SA; Hutter, D; Sismour, AM
Nucleic Acids Res. Suppl.3 125-126 (2003)
<Abstract>

Over 15 years ago, the Benner group noticed that the DNA alphabet need not be limited to the four standard nucleotides known in natural DNA. Rather, twelve nucleobases forming six base pairs joined by mutually exclusive hydrogen bonding patterns are possible within the geometry of the Watson-Crick pair (Fig. 1). Synthesis and studies on these compounds have brought us to the threshold of a synthetic biology, an artificial chemical system that does basic processes needed for life (in particular, Darwinian evolution), but with unnatural chemical structures. At the same time, the artificial genetic information systems (AEGIS) that we have developed have been used in FDA-approved commercial tests for managing HIV and hepatitis C infections in individual patients, and in a tool that seeks the virus for severe acute respiratory syndrome (SARS). AEGIS also supports the next generation of robotic probes to search for genetic molecules on Mars, Europa, and elsewhere where NASA probes will travel.

Act natural
Benner, SA
Nature421(6919) 118-118 (2003)

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins
Gaucher, EA; Thomson, JM; Burgan, MF; Benner, SA
Nature425(6955) 285-288 (2003)
<Abstract>

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences(1-9) of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65degreesC. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria(10), the distribution of thermophily in derived bacterial lineages(11), the inferred G+C content of ancient ribosomal RNA(12), and the geological record combined with assumptions concerning molecular clocks(13). The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Synthesis of 2-(3 '-azido- and 3 '-amino-3 '-deoxy-beta-D-ribofuranosyl)thiazole-4-carboxamide
Liang, CW; Kim, MJ; Jeong, LS; Chun, MW
Nuc. Nuc. Nuc. acids22(11), Taylor & Francis Group 2039-2048 (2003)
<Abstract>

In view of biological activities of tiazofurin and azido or aminosugar nucleosides, novel azido- and amino-substituted tiazofurin derivatives (I and 2) were efficiently synthesized starting from 1,2;5,6-di-O-isopropylidene-D-glucose.

C-5 modified nucleosides: Direct insertion of alkynyl-thio functionality in pyrimidines
Held, HA; Roychowdhury, A; Benner, SA
Nuc. Nuc. Nuc. acids22(4), Taylor & Francis Group 391-404 (2003)
<Abstract>

A route is presented to append, in a single step, alkynyl thioesters to the 5-position of a pyrimidine ring of a nucleoside that is unprotected. These products should be useful to support in vitro selection experiments with functionalized DNA.

Synthesis and biological evaluation of pyrimidine nucleosides fused with 3 ',4 '-tetrahydrofuran ring
Chun, MW; Kim, MJ; Kim, HO; Moon, HR; Kim, HD; Kim, JH; Jeong, LS
Nuc. Nuc. Nuc. acids22, Taylor & Francis Group 719-721 (2003)
<Abstract>

Pyrimidine nucleosides fused with 3,4'-tetrahydrofuran ring were synthesized, starting from 1,2;5,6-di-O-isopropylidene-D-glucose and assayed for antiviral activities. Thymine analogue 1 and its corresponding 2'-deoxy analogue 3 exhibited high cytotoxicity instead of giving antiviral activities.

Synthesis of 5-azacytidine nucleosides with rigid sugar moiety as potential antitumor agents
Chun, MW; Kim, MJ; Kim, HO; Kim, HD; Kim, JH; Moon, HR; Jeong, LS
Nuc. Nuc. Nuc. acids22, Taylor & Francis Group 915-917 (2003)
<Abstract>

The bicyclic 3'-O,5'-C-methylene-linked and 2'-O,5'-C-methylene-linked 5-azacytidine derivatives were readily synthesized from 1,2;5,6-di-O-isopropylidene-D-glucose and evaluated against several cancer cell lines.

Susceptible to intolerance - a range of hormonal actions in a susceptible Arabidopsis pathogen response
O'Donnell, PJ; Schmelz, EA; Moussatche, P; Lund, ST; Jones, JB; Klee, HJ
Plant J.33(2) 245-257 (2003)
<Abstract>

Ethylene and salicylic acid (SA) are key intermediates in a host's response to pathogens. Previously, we have shown using a tomato compatible interaction that ethylene and SA act sequentially and are essential for disease symptom production. Here, we have examined the relationship between the two signals in the Arabidopsis-Xanthomonas campestris pv. campestris (Xcc ) compatible interaction. Preventing SA accumulation by expression of the nahG gene reduced subsequent ethylene production and altered the development of disease symptoms, with plants showing no visible chlorosis. The ethylene insensitive lines, etr1-1 and etr2-1 , on the other hand, accumulated SA and exhibited normal but precocious symptom development. Therefore, Arabidopsis , like tomato, was found to exhibit co-operative ethylene and SA action for the production of disease symptoms. However, in Arabidopsis , SA was found to act upstream of ethylene. Jasmonic acid and indole-3-acetic acid levels were also found to increase in response to Xcc . In contrast to ethylene, accumulation of these hormones was not found to be dependent on SA action. These results indicate that the plants response to a virulent pathogen is a composite of multiple signaling pathways.

Nucleobase pairing in Watson-Crick-like genetic expanded information systems
Geyer, CR; Battersby, TR; Benner, SA
Structure11(12) 1485-1498 (2003)
<Abstract>

To guide the design of alternative genetic systems, we measured melting temperatures of DNA duplexes containing matched and mismatched nucleobase pairs from natural and unnatural structures. The pairs were analyzed in terms of structural features, including nucleobase size, number of hydrogen bonds formed, the presence of uncompensated hydrogen bonding functional groups, the nature of the bond joining the nucleobase to the sugar, and nucleobase charge. The results suggest that stability of nucleobase pairs correlates with the number of H-bonds, size complementarity, the presence of uncompensated functional groups, and the presence of charge on a nucleobase. Each of these properties appear to be more significant than the nature of the glycosidic bond and sequence context. The results provide guidelines for constructing stable Watson-Crick like nucleobase pairs with unnatural nucleobases. The experiments also demonstrate that expanded genetic systems can be constructed using size complementary nucleobase pairs that contain three hydrogen bonds.

1 -> 2 Migration and concurrent glycosidation of phenyl 1-thio-alpha-mannopyranosides via 2,3-O-cyclic dioxonium intermediates
Yang, ZY; Cao, HZ; Hu, J; Shan, RL; Yu, B
Tetrahedron59(2) 249-254 (2003)
<Abstract>

Treatment of phenyl 2,3-O-cyclic ketene acetal- and 2,3-O-thionocarbonyl-1-thio-mannopyranosides with TMSOTf and MeOTf, respectively, gave the corresponding 2,3-O-cyclic dioxonium intermediates, which proceeded via 1-->2 migration and concurrent glycosidation in the presence of alcohols to provide the corresponding 2-S-phenyl glycosides stereoselectively. While the former donors were too labile, the latter donors have proved superior for the present purpose. The X-ray crystallographic structures of phenyl 4-O-methyl-2,3-O-thiocarbonyl-1-thio-alpha-L-rhamnopyranoside (1), a typical donor for the present reaction, and its anomeric azide analogue (6), which could not undergo the present reaction under similar conditions, are provided. (C) 2002 Elsevier Science Ltd. All rights reserved.

Guanidine reduces stop codon read-through caused by missense mutations in SUP35 or SUP45
Bradley, ME; Bagriantsev, S; Vishveshwara, N; Liebman, SW
Yeast20(7) 625-632 (2003)
<Abstract>

Sup35 and Sup45 are essential protein components of the Saccharomyces cerevisiae ltranslation termination factor. Yeast cells harbouring the [PSI+] prion form of Sup35 have impaired stop codon recognition (nonsense suppression). It has long been known that the [PSI+] prion is not stably transmitted to daughter cells when yeast are grown in the presence of mm concentrations of guanidine hydrochloride (GuHCl). In this paper, Mendelian suppressor mutations whose phenotypes are likewise hidden during growth in the presence of millimolar GuHCl are described. Such GuHCl-remedial Mendelian suppressors were selected under conditions where [PSI+] appearance was limiting, and were caused by missense mutations in SUP35 or SUP45. Clearly, antisuppression caused by growth in the presence of GuHCl is not sufficient to distinguish missense mutations in SUP35 or SUP45, from [PSI+]. However, the Mendelian and prion suppressors can be distinguished by subsequent growth in the absence of GuHCl, where only the nonsense suppression caused by the [PSI+] prion remains cured. Recent reports indicate that GuHCl blocks the inheritance of [PSI+] by directly inhibiting the activity of the protein remodelling factor Hsp104, which is required for the transmission of [PSI+] from mother to daughter cells. However, the nonsense suppressor activity caused by the GuHCl-remedial sup35 or sup45 suppressors does not require Hsp104. Thus, GuHCl must antisuppress the sup35 and sup45 mutations via an in vivo target distinct from Hsp104. Copyright (C) 2003 John Wiley Sons, Ltd.

Phosphates, DNA, and the search for nonterrean life: A second generation model for genetic molecules
Benner, SA; Hutter, D
Bioorg. Chem.30(1) 62-80 (2002)
<Abstract>

Phosphate groups are found and used widely in biological chemistry. We have asked whether phosphate groups are likely to be important to the functioning of genetic molecules. including DNA and RNA. From observations made on synthetic analogs of DNA and RNA where the phosphates are replaced by nonanionic linking groups, we infer a set of rules that highlight the importance of the phosphodiester backbone for the proper functioning of DNA as a genetic molecule. The polyanionic backbone appears to give DNA the capability of replication following simple rules, and evolving. The polyanionic nature of the backbone appears to be critical to prevent the single strands from folding. permitting them to act as templates, guiding the interaction between two strands to form a duplex in a way that permits simple rules to guide the molecular recognition event, and buffering the sensitivity of its physicochemical properties to changes in sequence. We argue that the feature of a polyelectrolyte (polyanion or polycation) may be required for a "self-sustaining chemical system capable of Darwinian evolution." The polyelectrolyte structure therefore may be a universal signature of life, regardless of its genesis. and unique to living forms as well. (C) 2002 Elsevier Science (USA).

Complex glycosylation of Skp1 in Dictyostelium: implications for the modification of other eukaryotic cytoplasmic and nuclear proteins
West, CM; van der Wel, H; Gaucher, EA
Glycobiology12(2) (2002)
<Abstract>

Recently, complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the eukaryotic microorganism Dirtyostelium. Skp1's glycosylation is mediated by the sequential action of a prolyl hydroxylase and five conventional sugar nucleotide-dependent glycosyltransferase activities that reside in the cytoplasm rather than the secretory compartment. The Skp1-HyPro GlcNAc-Transferase, which adds the first sugar, appears to be related to a lineage of enzymes that originated in the prokaryotic cytoplasm and initiates mucin-type O-linked glycosylation in the lumen of the eukaryotic Golgi apparatus. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of beta1,3-linked Gal and alpha1,2-linked Fuc. The architecture of this enzyme resembles that of certain two-domain prokaryotic glycosyl-transferases. The catalytic domains are related to those of a large family of prokaryotic and eukaryotic, cytoplasmic, membrane-bound, inverting glycosyltransferases that modify glycolipids and polysaccharides prior to their translocation across membranes toward the secretory pathway or the cell exterior. The existence of these enzymes in the eukaryotic cytoplasm away from membranes and their ability to modify protein acceptors expose a new set of cytoplasmic and nuclear proteins to potential prolyl bydroxylation and complex O-linked glycosylation.

Identification of a Golgi-associated UDP-GlcNAc : polypeptide mucin-type alpha-N-acetylglucosaminyltransferase that modifies cell surface proteins in Dictyostelium
West, CM; van der Wel, H; Metcalf, T; Kaplan, L; Gaucher, EA
Glycobiology12(10) 697-697 (2002)

From phosphate to bis(methylene) sulfone: Non-ionic backbone linkers in DNA
Hutter, D; Blaettler, MO; Benner, SA
Helv. Chim. Acta85(9) 2777-2806 (2002)
<Abstract>

Chimeric DNA molecules containing four different linking groups, the natural phosphate, 5'-methylenephosphonate. bis(methylene)phosphinate, and bis(methylene) sulfone (see Fig.1), were directly compared for their ability to form duplexes with complementary DNA and DNA chimeras. From melting temperatures for analogous complementary sequences, general conclusions about the impact of geometric distortion of the internucleotide linkage around the two P-O-C bridges were drawn, as were conclusions about the impact on duplex stability that arises from the removal of the negative charge in the linking group. Each structural perturbation diminished the melting temperature, by ca. -2.5degrees per modification for the 5'-methylenephosphonate, -3.5degrees per modification for the bis(methylene)phosphinate, and -4.5degrees per modification for the bis(methylene) sulfone linker. These results have implications for DNA chemistry including the design of 'antisense' candidates and the proposal of alternative genetic materials in the search for non-terrean life.

Fourier transform-ion cyclotron resonance mass spectrometric resolution, identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library
Wigger, M; Eyler, JR; Benner, SA; Li, WQ; Marshall, AG
J. Am. Soc. Mass Spec.13(10) 1162-1169 (2002)
<Abstract>

The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent.

Detecting compensatory covariation signals in protein evolution using reconstructed ancestral sequences
Fukami-Kobayashi, K; Schreiber, DR; Benner, SA
J. Mol. Biol.319(3) 729-743 (2002)
<Abstract>

When protein sequences divergently evolve under functional constraints, some individual amino acid replacements that reverse the charge (e.g. Lys to Asp) may be compensated by a replacement at a second position that reverses the charge in the opposite direction (e.g. Glu to Arg). When these side-chains are near in space (proximal), such double replacements might be driven by natural selection, if either is selectively disadvantageous, but both together restore fully the ability of the protein to contribute to fitness (are together "neutral"). Accordingly, many have sought to identify pairs of positions in a protein sequence that suffer compensatory replacements, often as a way to identify positions near in space in the folded structure. A "charge compensatory signal" might manifest itself in two ways. First, proximal charge compensatory replacements may occur more frequently than predicted from the product of the probabilities of individual positions suffering charge reversing replacements independently. Conversely, charge compensatory pairs of changes may be observed to occur more frequently in proximal pairs of sites than in the average pair. Normally, charge compensatory covariation is detected by comparing the sequences of extant proteins at the "leaves" of phylogenetic trees. We show here that the charge compensatory signal is more evident when it is sought by examining individual branches in the tree between reconstructed ancestral sequences at nodes in the tree. Here, we find that the signal is especially strong when the positions pairs are in a single secondary structural unit (e.g. ut helix or P strand) that brings the side-chains suffering charge compensatory covariation near in space, and may be useful in secondary structure prediction. Also, "node-node" and "node-leaf" compensatory covariation may be useful to identify the better of two equally parsimonious trees, in a way that is independent of the mathematical formalism used to construct the tree itself. Further, compensatory covariation may provide a signal that indicates whether an episode of sequence evolution contains more or less divergence in functional behavior. Compensatory covariation analysis on reconstructed evolutionary trees may become a valuable tool to analyze genome sequences, and use these analyses to extract biomedically useful information from proteome databases. (C) 2002 Elsevier Science Ltd. All rights reserved.

Oligodeoxyribonucleotide analogues with bridging dimethylene sulfide, sulfoxide, and sulfone groups. Toward a second-generation model of nucleic acid structure
Huang, Z; Benner, SA
J. Org. Chem.67(12) 3996-4013 (2002)
<Abstract>

Short DNA analogues with bridging dimethylene sulfide, sulfoxide, and sulfone groups replacing the phosphate diesters (S-DNAs) were synthesized from building blocks prepared via two routes, both starting from D-glucose. Building blocks for RNA analogues were prepared by stereoselective introduction of nucleobase into a 2'-acylated ribose analogue. The ribose analogues were converted to deoxyribose analogues by replacement of a 3"-OH group by a thioacetyl unit, followed by photolytic deoxygenation or radical-based 2'-deoxygenation. DNA analogues joined via CH2-S-CH2 units were prepared by S(N)2 displacement of a 6'-mesyl group on one building block using a thiolate nucleophile of another. 4,4'-Dimethoxytrityl protection and deprotection schemes were established for both the thiol and hydroxyl groups. The corresponding sulfoxide DNA analogues were obtained by oxidation with hydrogen peroxide. Sulfone DNA analogues were obtained by oxidation of the sulfide DNA with persulfate or hydrogen peroxide in the presence of a titanium silicate catalyst. The physical properties of several representative oligonucleotide analogues were examined, and interpreted in light of a "second-generation" model for DNA strand-strand recognition, a model that emphasizes the role of the polyanionic backbone in diminishing unwanted tendencies of highly functionalized molecules to form "structure" in solution. Even short sulfide-linked DNA analogues displayed,association properties different from those displayed by standard DNA molecules. Complex formation observed with sulfide-linked tetramers by HPLC study in different solvents suggested that the complex is formed using hydrogen bonding. Sulfone-linked dinucleotides display Watson-Crick behavior; the tetramer, however, displayed self-structure. Self-structure and self-aggregation become more prominent as the length of the oligonucleotide analogues increases. The tendency to self-aggregate can be decreased by adding a charged sulfonate group to the 3"-end of the DNA analogue. Features of the second-generation model are important for many areas of nucleic acid chemistry, from the design of nucleic acid therapeutic agents to the search for life on other planets.

Evolution of amino acid frequencies in proteins over deep time: Inferred order of introduction of amino acids into the genetic code.
Brooks, DJ; Fresco, JR; Lesk, AM; Singh, M
Mol. Biol. Evol.19 1645-1655 (2002)
<Abstract>

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.

The crystal structure of eEF1A refines the functional predictions of an evolutionary analysis of rate changes among elongation factors
Gaucher, EA; Das, UK; Miyamoto, MM; Benner, SA
Mol. Biol. Evol.19(4) 569-573 (2002)

Increased frequency of cysteine, tyrosine and phenylalanine residues since the Last Universal Ancestor.
Brooks, DJ; Fresco, JR
Mol. Cell. Proteomics1 125-131 (2002)
<Abstract>

Analysis of extant proteomes has the potential of revealing how amino acid frequencies within proteins have evolved over biological time. Evidence is presented here that cysteine, tyrosine, and phenylalanine residues have substantially increased in frequency since the three primary lineages diverged more than three billion years ago. This inference was derived from a comparison of amino acid frequencies within conserved and non-conserved residues of a set of proteins dating to the last universal ancestor in the face of empirical knowledge of the relative mutability of these amino acids. The under-representation of these amino acids within last universal ancestor proteins relative to their modern descendants suggests their late introduction into the genetic code. Thus, it appears that extant ancient proteins contain evidence pertaining to early events in the formation of biological systems.

Challenging artificial genetic systems: thymidine analogs with 5-position sulfur functionality
Held, HA; Benner, SA
Nucl. Acids Res.30(17) 3857-3869 (2002)
<Abstract>

Eight different polymerases, chosen from evolutionary families A (Taq, Tfl, HotTub and Tth) and B (Pfu, Pwo, Vent and Deep Vent), were examined for their ability to incorporate 5-position modified 2'-deoxyuridine derivatives that carry a protected thiol group appended via different linkers containing either three or four carbon atoms. This represents the first attempt to incorporate the thiol functionality into DNA via enzymatic synthesis. Each polymerase-substrate combination was evaluated using a hierarchy of increasingly more difficult challenges, starting with incorporation of a single derivative, proceeding to incorporation of two derivatives at adjacent sites and non-adjacent sites, then examining the ability of the polymerase to accept the derivative within the template, and concluding with a challenge involving PCR. The evaluation of thiol-bearing 2'-deoxyuridine derivatives was then extended to consider their chemical stabilities. Stability was found to be less than satisfactory when the thiol functionality has a 'propargylic' relationship to the unsaturation in the linker. The best polymerase-appendage combination used the polymerase from Pyrococcus woesei (Pwo) and the 5'-tBu-SS-CH2-CH2-Cequivalent toC- linker. This pair supported PCR amplification and therefore should have value in artificial in vitro selection experiments. Indeed, we discovered that Pwo and Pfu preferred the derivative triphosphate over TTP, the natural substrate, in competition studies. These studies confirm an earlier suggestion that membership of an evolutionary family of polymerases is a partial predictor of the ability of the polymerase to accept 5-modified 2'-deoxyuridines. Considerable differences are displayed by different members within a polymerase family, however. This remains curious, as the ability of the polymerase to replicate natural DNA with high fidelity and its propensity to exclude unnatural analogs are presumed to be correlated.

Interactions among prions and prion "strains" in yeast
Bradley, ME; Edskes, HK; Hong, JY; Wickner, RB; Liebman, SW
Proc. Natl. Acad. Sci. USA99(7) 16392-16399 (2002)
<Abstract>

Prions are "infectious" proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PSI+] prion form its function is compromised. When Rnq1 is aggregated in its [PIN+] prion form, it promotes the de novo appearance of [PSI+]. Heritable variants (strains) of [PSI+] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PIN+] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PSI+]. Unlike [PSI+] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PIN+] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PSI+], or different [PIN+] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PIN+] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PSI+] appearance, [PIN+] enhances the de novo appearance of [URE3]. Surprisingly, [PSI+] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.

The past as the key to the present: Resurrection of ancient proteins from eosinophils
Benner, SA
Proc. Natl. Acad. Sci. USA99(8) 4760-4761 (2002)

Evolution - Planetary biology - Paleontological, geological, and molecular histories of life
Benner, SA; Caraco, MD; Thomson, JM; Gaucher, EA
Science296(5569) 864-868 (2002)
<Abstract>

The history of life on Earth is chronicled in the geological strata, the fossil record, and the genomes of contemporary organisms. When examined together, these records help identify metabolic and regulatory pathways, annotate protein sequences, and identify animal models to develop new drugs, among other features of scientific and biomedical interest. Together, planetary analysis of genome and proteome databases is providing an enhanced understanding of how life interacts with the biosphere and adapts to global change.

Ribonucleases in ruminants - Response
Benner, SA
Science297(5584) 1122-1122 (2002)

Predicting functional divergence in protein evolution by site-specific rate shifts
Gaucher, EA; Gu, X; Miyamoto, MM; Benner, SA
Trends Biochem. Sci.27(6) 315-321 (2002)
<Abstract>

Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the 'non-homogeneous gamma model', which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

Nucleosides and nucleotides. 208. alternate-strand triple-helix formation by the 3 '-3 '-linked oligodeoxynucleotides with the anthraquinonyl group at the junction point
Ueno, Y; Mikawa, M; Hoshika, S; Matsuda, A
Bioconjugate Chem.12(4) 635-642 (2001)
<Abstract>

The synthesis of 3 ' -3 ' -linked oligodeoxynucleotides (ODNs) with the anthraquinonyl group at the junction point is described. The ODNs were synthesized on a DNA synthesizer using a controlled pore glass (CPG) carrying pentaerythritol that has an intercalator at one of the four hydroxymethyl groups. Stability of the triplexes with the target duplexes was studied by thermal denaturation. The 3 ' -3 ' -linked ODNs with the anthraquinonyl group enhanced the thermal stability of the triplexes when compared with those without the intercalator and the unmodified nonamer 10. It was found that the ODNs 12 and 13 carrying the anthraquinonyl groups can form thermally stable triplexes by skipping two or three extra base pairs between two binding domains of the target duplexes. The ability of the 3 ' -3 ' -linked ODNs to inhibit cleavage of the target DNA 22 by the restriction enzyme Hind III was tested. It was found that the 3 ' -3 ' -linked ODN 16 with the anthraquinonyl group at the junction point inhibited the cleavage by the enzyme more effectively than the nonamer 14 and the 3 ' -3 ' -linked ODN 15 without the intercalator.

Stereoselective synthesis of 2-S-ethyl(phenyl)-2-thio-beta-glucopyranosides via 1,2-migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-mannopyranosides
Yang, ZY; Yu, B
Carbohydr. Res.333(2) 105-114 (2001)
<Abstract>

1,2-Migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha -D- and L-mannopyranosides under the action of TMSOTf readily afforded the corresponding 2-S-ethyl(phenyl)-2-thio-beta -glucopyranosides ready precursors to 2-deoxy-arabino-hexopyranosides (2-deoxy-beta -glucopyranosides). (C) 2001 Elsevier Science Ltd. All rights reserved.

Prions affect the appearance of other prions: The story of [PIN+]
Derkatch, IL; Bradley, ME; Hong, JY; Liebman, SW
Cell106(2) 171-182 (2001)
<Abstract>

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN+] is required for the de novo appearance of the [PSI+] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN+]. Thus, [PIN+] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN+] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.

Solution structure of the mEGF/TGF alpha(44-50) chimeric growth factor
Chamberlin, SG; Brennan, L; Puddicombe, SM; Davies, DE; Turner, DL
Euro. J. Biochem.268(23) 6247-6255 (2001)
<Abstract>

The solution structure of the growth factor chimera mEGF/TGF alpha (44-50) has been determined using an extended version of the DYANA procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H-bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site-specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero- and homodimerization of the c-erbB receptors.

A bifunctional diglycosyltransferase forms the Fuca1,2Galb,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium
van der Wel, H; Fisher, SZ; Gaucher, EA; West, CM
Glycobiology11(10) 884-884 (2001)

A stable dirhodium tetracarboxylate carbenoid: Crystal structure, bonding analysis, and catalysis
Snyder, JP; Padwa, A; Stengel, T; Arduengo, AJ; Jockisch, A; Kim, HJ
J. Am. Chem. Soc.123(45) 11318-11319 (2001)

Functional genomic, biochemical and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene
Johnson, CLV; Pechonick, EM; Park, SD; Havemann, GD; Leal, NA; Bobik, TA
J. Bacteriol.183 1577-1584 (2001)
<Abstract>

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.

PTC effect of carbon black filled polypropylene
He, XJ; Chen, F; Chen, XF
J. Mater. Sci. Lett.20(7) 589-590 (2001)

Fluorescent charge-neutral analogue of xanthosine: Synthesis of a 2 '-deoxyribonucleoside bearing a 5-aza-7-deazaxanthine base
Rao, P; Benner, SA
J. Org. Chem.66(15) 5012-5015 (2001)
<Abstract>

A concise route is described to prepare the 5-aza-7-deazapurine 2 ' -deoxyriboside (4), which presents the puADA hydrogen-bonding pattern, analogous to the hydrogen-bonding pattern presented by 2 ' -deoxyxanthosine (2). The route begins with the commercially available 1-alpha -chloro-2-deoxy-3-5-bistoluoyloxyribofuranose (10), which proves to be a versatile point of entry to beta -2 ' -deoxyribofuranosides. In the first step, 2-nitroimidazole (8) is coupled with 10 to yield intermediate 11. Reduction of the nitro group to an amino group yields 12, which is treated with phenyl isocyanatoformate to complete the nucleobase to yield 13. Removal of the toluoyloxy protecting groups of 13 yields the target nucleoside 4 in 40% overall yield in four steps. In an alternative strategy, convergent coupling of 14 with 10 under basic conditions was attempted but found to yield the heterocycle glycosylated at the undesired position. Compound 13 displays potentially useful fluorescence properties. After excitation at 250 nm, a solution of 13 in MeCN shows a fluorescence emission with a maximum at 410 Dm. Furthermore, 13 is neutral at physiological pH, a property that it shares with natural nucleobases but not xanthosine itself, which is an acid with a pK(a) of ca. 5.6. Furthermore, as part of the design, 4 is made capable of presenting an unshared pair of electrons to the DNA minor groove.

Natural progression
Benner, SA
Nature409(6819) 459-459 (2001)

Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations
Xu, YZ; Karalkar, NB; Kool, ET
Nat. Biotechnol.19(2) 148-152 (2001)
<Abstract>

Enzymatic ligation methods are useful in diagnostic detection of DNA sequences. Here we describe the investigation of nonenzymatic phosphorothioate-iodide DNA autoligation chemistry as a method for detection and identification of both RNA and DNA sequences. Combining ligation specificity with the hybridization specificity of the ligated product is shown to yield discrimination of a point mutation as high as >10(4)-fold. Unlike enzymatic ligations, this reaction is found to be equally efficient on RNA or DNA templates. The reaction is also shown to exhibit a significant level of self-amplification, with the template acting in catalytic fashion to ligate multiple pairs of probes. A strategy for fluorescence labeling of three autoligating energy transfer (ALET) probes and directly competing them for autoligation on a target sequence is described. The method is tested in several formats, including solution phase, gel, and blot assays. the ALET probe design offers direct RNA detection, combining high sequence specificity with an easily detectable color change by fluorescence resonance energy transfer (FRET).

Synthesis of novel 3 '-deoxy-3 '-C-hydroxymethyl nucleosides with conformationally rigid sugar moiety as potential antiviral agents
Chun, MW; Kim, MJ; Jo, UH; Kim, JH; Kim, HD; Jeong, LS
Nuc. Nuc. Nuc. acids20, Taylor & Francis Group 699-702 (2001)
<Abstract>

Based on the fact that the ring expanded 3'-C-hydroxymethyl analogue of oxetanocin A exhibited potent antiviral activity, two types of conformationally rigid 3'-C-hydroxymethyl derivatives in which 2'-hydroxyl group is linked to the 4'-position or to the 6'-position were synthesized starting from 1,2;5,6-di-O-isopropylidene-D-glucose, respectively.

Synthesis of novel D- and L-3 '-deoxy-3 '-C-hydroxymethyl nucleoside with exocyclic methylene as potential ribonucleotide reductase inhibitor
Chun, MW; Kim, MJ; Jo, UH; Kim, JH; Kim, HD; Jeong, LS
Nuc. Nuc. Nuc. acids20, Taylor & Francis Group 703-706 (2001)
<Abstract>

D- and L-3'-Deoxy-3'-C-hydroxymethyl thymidine substituted with exocyclic methylene at 2'-position were synthesized, starting from D- and L-xylose as potential ribonucleotide reductase inhibitor, respectively, but they were found to be inactive against several tumor cell lines.

Stereoselective synthesis of 2-S-phenyl-2-deoxy-beta-glycosides using phenyl 2,3-O-thionocarbonyl-1-thioglycoside donors via 1,2-migration and concurrent glycosidation
Yu, B; Yang, ZY
Org. Lett.3(3) 377-379 (2001)
<Abstract>

[GRAPHICS] 1,2-Migration and concurrent glycosidation of phenyl 2,3-O-thionocarbonyl 1-thio-alpha -L-rhamnopyranosides under the action of methyl trifluoromethanesulfonate (MeOTf) afforded in high yields the 3-O-(methylthio)carbonyl-2-S-phenyl-2,6-dideoxy-beta -L-glucopyranosides ready precursors to the corresponding 2-deoxy-beta -glycosides.

Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors
Gaucher, EA; Miyamoto, MM; Benner, SA
Proc. Natl. Acad. Sci. USA98(2) 548-552 (2001)
<Abstract>

The divergent evolution of protein sequences from genomic databases can be analyzed by the use of different mathematical models. The most common treat all sites in a protein sequence as equally variable. More sophisticated models acknowledge the fact that purifying selection generally tolerates variable amounts of amino acid replacement at different positions in a protein sequence. In their "stationary" versions, such models assume that the replacement rate at individual positions remains constant throughout evolutionary history. "Nonstationary" covarion versions, however, allow the replacement rate at a position to vary in different branches of the evolutionary tree. Recently, statistical methods have been developed that highlight this type of variation in replacement rates. Here, we show how positions that have variable rates of divergence in different regions of a tree ("covarion behavior"), coupled with analyses of experimental three-dimensional structures, can provide experimentally testable hypotheses that relate individual amino acid residues to specific functional differences in those branches. We illustrate this in the elongation factor family of proteins as a paradigm for applications of this type of analysis in functional genomics generally.

Evolution, language and analogy in functional genomics
Benner, SA; Gaucher, EA
Trends in Genetics17(7) 414-418 (2001)
<Abstract>

Almost a century ago, Wittgenstein pointed out that theory in science is intricately connected to language. This connection is not a frequent topic in the genomics literature. But a case can be made that functional genomics is today hindered by the paradoxes that Wittgenstein identified. If this is true, until these paradoxes are recognized and addressed, functional genomics will continue to be limited in its ability to extrapolate information from genomic sequences.

Beyond BLAST: Paleogenomics tools to infer function to genetic sequences.
Benner, S; Chamberlin, S
Am. J. Hum. Genet.67(4) 260-260 (2000)

X-ray crystallographic and analytical ultracentrifugation analyses of truncated and full-length yeast copper chaperones for SOD (LYS7): A dimer-dimer model of LYS7-SOD association and copper delivery
Hall, LT; Sanchez, RJ; Holloway, SP; Zhu, HN; Stine, JE; Lyons, TJ; Demeler, B; Schirf, V; Hansen, JC; Nersissian, AM; Valentine, JS; Hart, PJ
Biochemistry39(13), ACS 3611-3623 (2000)
<Abstract>

Copper-zinc superoxide dismutase (CuZnSOD) acquires its catalytic copper ion through interaction with another polypeptide termed the copper chaperone for SOD. Here, we combine X-ray crystallographic and analytical ultracentrifugation methods to characterize rigorously both truncated and full-length forms of apo-LYS7, the yeast copper chaperone for SOD. The 1.55 Angstrom crystal structure of LYS7 domain 2 alone (L7D2) was determined by multiple-isomorphous replacement (MIR) methods. The monomeric structure reveals an eight-stranded Greek key beta-barrel similar to that found in yeast CuZnSOD, but it is substantially elongated at one end where the loop regions of the beta-barrel come together to bind a calcium ion. In agreement with the crystal structure, sedimentation velocity experiments indicate that L7D2 is monomeric in solution under all conditions and concentrations that were tested. In contrast, sedimentation velocity and sedimentation equilibrium experiments show that full-length apo-LYS7 exists in a monomer-dimer equilibrium under nonreducing conditions. This equilibrium is shifted toward the dimer by approximately 1 order of magnitude in the presence of phosphate anion. Although the basis for the specificity of the LYS7-SOD interaction as well as the exact mechanism of copper insertion into SOD is unknown, it has been suggested that a monomer of LYS7 and a monomer of SOD may associate to form a heterodimer via L7D2. The data presented here, however, taken together with previously published crystallographic and analytical gel filtration data on full-length LYS7, suggest an alternative model wherein a dimer of LYS7 interacts with a dimer of yeast CuZnSOD. The advantages of the dimer-dimer model over the heterodimer model are enumerated.

Rearrangement of sugar 1,2-orthoesters to glycosidic products: a mechanistic implication
Yang, ZY; Lin, WB; Yu, B
Carbohydr. Res.329(4) 879-884 (2000)
<Abstract>

Dependence and independence of [PSI+] and [PIN+]: A two-prion system in yeast?
Derkatch, IL; Bradley, ME; Masse, SVL; Zadorsky, SP; Polozkov, GV; Inge-Vechtomov, SG; Liebman, SW
EMBO J.19(9) 1942-1952 (2000)
<Abstract>

The [PSI+] prion can be induced by overproduction of the complete Sup35 protein, but only in strains carrying the non-Mendelian [PIN+] determinant. Here we demonstrate that just as [psi(-)] strains can exist as [PIN+] and [pin(-)] variants, [PSI+] can also exist in the presence or absence of [PIN+]. [PSI+] and [PIN+] tend to be cured together, but can be lost separately. [PSI+]-related phenotypes are not affected by [PIN+]. Thus, [PIN+] is required for the de nora formation of [PSI+], not for [PSI+] propagation. Although [PSI+] induction is shown to require [PIN+] even when the only overexpressed region of Sup35p is the prion domain, two altered prion domain fragments circumventing the [PIN+] requirement are characterized. Finally, in strains cured of [PIN+], prolonged incubation facilitates the reappearance of [PIN+]. Newly. appearing [PIN+] elements are often unstable but become stable in some mitotic progeny. Such reversibility of curing, together with our previous demonstration that the inheritance of [PIN+] is non-Mendelian, supports the hypothesis that [PIN+] is a prion, Models for [PIN+] action, which explain these findings, are discussed.

Synthesis and characterization of oligonucleotides containing 2 '-deoxyxanthosine using phosphoramidite chemistry
Jurczyk, SC; Horlacher, J; Devined, KG; Benner, SA; Battersby, TR
Helv. Chim. Acta83(7) 1517-1524 (2000)
<Abstract>

Oligodeoxynucleotides containing 2'-deoxyxanthosine (X-d) were synthesized in good yield from a O-2,O-6-bis[2-(4-nitrophenyl)ethyl](NPE)-protected phosphoramidite of X-d. Attempts to synthesize a O-6-monoNPE-protected phosphoramidite resulted in formation of a major by-product. The NPE protecting groups were removed by treatment with oximate ion after other protecting groups were removed with aqueous NH,OH solution. The composition of the synthetic oligonucleotides was verified by enzymatic degradation and MALDI-TOF mass spectrometry. The efficacy of this procedure allowed isolation of oligodeoxynucleotides containing multiple X-d residues.

The copper transport protein Atox1 promotes neuronal survival
Kelner, GS; Lee, MH; Clark, ME; Maciejewski, D; McGrath, D; Rabizadeh, S; Lyons, T; Bredesen, D; Jenner, P; Maki, RA
J. Biol. Chem.275(1), ASBMB 580-584 (2000)
<Abstract>

Atox1, a copper transport protein, was recently identified as a copper dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.

The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis
Lyons, TJ; Nersissian, A; Huang, HJ; Yeom, H; Nishida, CR; Graden, JA; Gralla, EB; Valentine, JS
J. Biol. Inorg. Chem.5(2) 189-203 (2000)
<Abstract>

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pK(a). The mutants H71C and D83A did not bind Co2+ in a wildtype-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Evaluation measures of multiple sequence alignments
Gonnet, GH; Korostensky, C; Benner, S
J. Comp. Bio.7 261-276 (2000)
<Abstract>

Multiple sequence alignments (MSAs) are frequently used in the study of families of protein sequences or DNA/RNA sequences. They are a fundamental tool for the understanding of the structure, functionality and, ultimately, the evolution of proteins. A new algorithm, the Circular Sum (CS) method, is presented for formally evaluating the quality of an MSA, It is based on the use of a solution to the Traveling Salesman Problem, which identifies a circular tour through an evolutionary tree connecting the sequences in a protein family. With this approach, the calculation of an evolutionary tree and the errors that it mould introduce can be avoided altogether, The algorithm gives an upper bound, the best score that can possibly be achieved by any MSA for a given set of protein sequences. Alternatively, if presented with a specific MSA, the algorithm provides a formal score for the MSA, which serves as an absolute measure of the quality of the MSA, The CS measure yields a direct connection between an MSA and the associated evolutionary tree, The measure can be used as a tool for evaluating different methods for producing MSAs, A brief example of the last application is provided, Because it weights all evolutionary events on a tree identically, but does not require the reconstruction of a tree, the CS algorithm has advantages over the frequently used sum-of-pairs measures for scoring MSAs, which weight some evolutionary events more strongly than others. Compared to other weighted sum-of-pairs measures, it has the advantage that no evolutionary tree must be constructed, because we can find a circular tour without knowing the tree.

Evolutionary history of the uterine serpins
Peltier, MR; Raley, LC; Liberles, DA; Benner, SA; Hansen, PJ
J. Exp. Zoo.288(2) 165-174 (2000)
<Abstract>

A bioinformatics analysis was conducted on the four members of the uterine serpin (US) family of serpins. Evolutionary analysis of the protein sequences and 86 homologous serpins by maximum parsimony and distance methods indicated that the uterine serpins proteins form a clade distinct from other serpins. Ancestral sequences were reconstructed throughout the evolutionary tree by parsimony. These suggested that some branches suffered a high ratio of nonsynonymous to synonymous mutations, suggesting episodes of adaptive evolution within the serpin family. Analysis of the sequences by neutral evolutionary distance methods suggested that the uterine serpins diverged from other serpins prior to the divergence of the mammals from other vertebrates. The porcine uterine serpins are paralogs that diverged from a single common ancestor within the Sus genus after pigs separated from other artiodactyls. The uterine serpins contain several protein kinase C and tyrosine kinase phosphorylation sites. These sites may be important for the lymphocyte-inhibitory activity of OvUS if, Like other basic proteins, OvUS can cross the cell membrane of an activated lymphocyte. Internalized OvUS could serve as an alternative target to protein kinases important for the mitogenic response to antigens. (C) 2000 Wiley-Liss, Inc.

A statistical view of FMRFamide neuropeptide diversity
Espinoza, E; Carrigan, M; Thomas, SG; Shaw, G; Edison, AS
Mol. Neurobiol.21 35-56 (2000)
<Abstract>

FMRFamide-like peptide (FLP) amino acid sequences have been collected and statistically analyzed. FLP amino acid composition as a function of position in the peptide is graphically presented for several major phyla. Results of total amino acid composition and frequencies of pairs of FLP amino acids have been computed and compared with corresponding values from the entire GenBank protein sequence database. The data for pairwise distributions of amino acids should help in future structure-function studies of FLPs. To aid in future peptide discovery, a computer program and search protocol was developed to identify FLPs from the GenBank protein database without the use of keywords.

The missing organic molecules on Mars
Benner, SA; Devine, KG; Matveeva, LN; Powell, DH
Proc. Natl. Acad. Sci. USA97(6) 2425-2430 (2000)
<Abstract>

GC-MS on the Viking 1976 Mars missions did not detect organic molecules on the Martian surface, even those expected from meteorite bombardment. This result suggested that the Martian regolith might hold a potent oxidant that converts all organic molecules to carbon dioxide rapidly relative to the rate at which they arrive. This conclusion is influencing the design of Mars missions. We reexamine this conclusion in light of what is known about the oxidation of organic compounds generally and the nature of organics likely to come to Mars via meteorite. We conclude that nonvolatile salts of benzenecarboxylic acids, and perhaps oxalic and acetic acid, should be metastable intermediates of meteoritic organics under oxidizing conditions. Salts of these organic acids would have been largely invisible to GC-MS, Experiments show that one of these, benzenehexacarboxylic acid (mellitic acid), is generated by oxidation of organic matter known to come to Mars, is rather stable to further oxidation, and would not have been easily detected by the Viking experiments. Approximately 2 kg of meteorite-derived mellitic acid may have been generated per m(2) of Martian surface over 3 billion years. How much remains depends on decomposition rates under Martian conditions, As available data do not require that the surface of Mars be very strongly oxidizing, some organic molecules might be found near the surface of Mars, perhaps in amounts sufficient to be a resource. Missions should seek these and recognize that these complicate the search for organics from entirely hypothetical Martian life.

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast
Lyons, TJ; Gasch, AP; Gaither, LA; Botstein, D; Brown, PO; Eide, DJ
Proc. Natl. Acad. Sci. USA97(14) 7957-7962 (2000)
<Abstract>

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Functional inferences from reconstructed evolutionary biology involving rectified databases. An evolutionarily-grounded approach to functional genomics.
Benner, SA; Chamberlin, SG; Liberles, DA; Govindarajan, S; Knecht, L
Res. MicroBiol.151(2) 97-106 (2000)
<Abstract>

If bioinformatics tools are constructed to reproduce the natural, evolutionary history of the biosphere, they offer powerful approaches to some of the most difficult tasks in genomics, including the organization and retrieval of sequence data, the updating of massive genomic databases, the detection of database error, the assignment of introns, the prediction of protein conformation from protein sequences, the detection of distant homologs, the assignment of function to open reading frames, the identification of biochemical pathways from genomic data, and the construction of a comprehensive model correlating the history of biomolecules with the history of planet Earth.

Unite efforts and conquer mysteries of artificial genetics
Benner, SA
Science290(5496) 1506-1506 (2000)

A novel and expeditious approach to the stereoselective synthesis of 2-S-ethyl(phenyl)-2-deoxy-beta-glycosides, ready precursors to 2-deoxy-beta-glycosides
Yu, B; Yang, ZY
Tet. Lett.41(16) 2961-2964 (2000)
<Abstract>

1,2-Migration and concurrent glycosidation of ethyl(phenyl) 2,3-orthoester-1-thio-alpha-1-rhamnopyranosides under the action of TMSOTf readily afforded the corresponding 2-S-ethyl(phenyl)-2-deoxy-beta-glycosides, ready precursors to 2-deoxy-beta-glycosides. (C) 2000 Elsevier Science Ltd. All rights reserved.

Evolutionary, mechanistic, and predictive analyses of the hydroxymethyldihydropterin pyrophosphokinase family of proteins
Gerloff, DL; Cannarozzi, GM; Joachimiak, M; Cohen, FE; Schreiber, D; Benner, SA
Biochem. Biophys. Res. Comm.254(1) 70-76 (1999)
<Abstract>

A prediction has been prepared ab initio for the secondary structure of the hydroxymethyldihydropterin pyrophosphokinase (HPPK) family of proteins starting from a set of aligned homologous protein sequences. Attempts to identify a fold by threading failed, judging; by the inability to iind a threading "hit" that had a secondary structure that was plausibly congruent to the predicted secondary structure for the HPPK, family. Therefore, a set of tertiary structure models was assembled ab initio, where alternative models were built and used to select between alternative secondary structure models. This prediction report illustrates the importance of non-computational approaches to structure prediction at its present frontier, which is to obtain medium resolution models of tertiary structure. (C) 1999 Academic Press.

Simple one-pot synthesis of a 2 '-tritium labeled C-deoxynucleoside
Lutz, S; Benner, SA
Bioorg. Med. Chem. Lett.9(5) 723-726 (1999)
<Abstract>

The deoxygenation and 2'-labeling of a C-ribonucleoside by reductive elimination with tri-n-butyltin hydride[H-3] in a one-pot reaction is described. The approach is a safe, simple, efficient, and general method for 2'-labeling of nucleosides. (C) 1999 Elsevier Science Ltd. All rights reserved.

Sequence analysis of FMRFamide-like peptides and precursors
Carrigan, M; Espinoza, E; Thomas, S; Benner, SA; Edison, AS
Brain Res.848 (1999)

Structural changes to ribonuclease A and their effects on biological activity
Soucek, J; Raines, RT; Haugg, M; Raillard-Yoon, SA; Benner, SA
Comp. Biochem. Phys. C123(2) 103-111 (1999)
<Abstract>

Bovine seminal ribonuclease (BS RNase) displays immunosuppressive and antitumor activities on mammalian cells, whereas bovine pancreatic ribonuclease (RNase A) is not cytotoxic. To learn more about the mechanism of BS RNase cytotoxicity, various mutants and hybrid proteins were prepared. A series of RNase A variants substituted with amino acid residues from BS RNase were prepared. Concerning quaternary structure, a significant impact was achieved in the variant TM (Q28L K31C S32C), which forms a dimer joined covalently by two intersubunit disulfide bonds. This variant is more efficient than RNase A but less active than BS RNase. Introduction of cationic residues at positions 55, 62, and 64 or substitution at positions 111 and 113 enhanced the immunosuppressive activity of RNase A but did not confer its antitumor activity. The substitution at positions 28, 31, 32, 55, 62, 64, 111, and 113 in variant T13 exerted the best immunosuppressive and antitumor effect observed among the round of the RNase A variants. Replacement of the active-site histidine residues H12 and H119 with asparagine led to the loss of both catalytic and biological activities. Five previously prepared hybrid enzymes (SRA 1-5), synthesized by introducing 16 amino acid residues from RNase A into BS RNase, exerted the same immunosuppressive activities as did the wild-type BS RNase. However, the substitution at positions 111, 113, and 115 in variant SRA 5 caused a marked decrease in its antitumor effect, indicating that these residues play an important role in antitumor efficiency. A different mechanism of action of RNases on tumor cells and/or on blastogenic transformed lymphocytes has been assumed. (C) 1999 Elsevier Science Inc. All rights reserved.

The PNM2 mutation in the prion protein domain of SUP35 has distinct effects on different variants of the [PSI+] prion in yeast
Derkatch, IL; Bradley, ME; Zhou, P; Liebman, SW
Curr. Genet.35(2) 59-67 (1999)
<Abstract>

We have previously described different variants of the yeast prion [PSI+] that can be obtained and maintained in the same genetic background. These [PSI+] variants, which differ in the efficiency of nonsense suppression, mitotic stability and the efficiency of curing by GuHCl, may correspond to different [PSI+] prion conformations of Sup35p or to different types of prion aggregates. Here we investigate the effects of overexpressing a mutant allele of SUP35 and find different effects on weak and strong [PSI+] variants: the suppressor phenotype of weak [PSI+] factors is increased, whereas the suppressor effect of strong [PSI+] factors is reduced. The SUP35 mutation used was originally described as a "Psi no more" mutation (PNM2) because it caused loss of [PSI+]. However, none of the [PSI+] variants in the strains used in our study were cured by PNM2. Indeed, when overexpressed, PNM2 induced the de novo appearance of both weak and strong [PSI+] variants with approximately the same efficiency as the overexpressed wild-type SUP35 allele. Our data suggest that the change in the region of oligopeptide repeats in the Sup35p N-terminus due to the PNM2 mutation modifies, but does not impair, the function of the prion domain of Sup35p.

Crystal structure of a hybrid between ribonuclease A and bovine seminal ribonuclease - the basic surface, at 2.0 angstrom resolution
Vatzaki, EH; Allen, SC; Leonidas, DD; Trautwein-Fritz, K; Stackhouse, J; Benner, SA; Acharya, KR
Euro. J. Biochem.260(1) 176-182 (1999)
<Abstract>

variant of bovine pancreatic ribonuclease A has been prepared with seven amino acid substitutions (Q55K, N62K, A64T, Y76K, S80RI E111G, N113K). These substitutions recreate in RNase A the basic surface found in bovine seminal RNase, a homologue of pancreatic RNase that diverged some 35 million years ago. Substitution of a portion of this basic surface (positions 55, 62, 64, 111 and 113) enhances the immunosuppressive activity of the RNase variant, activity found in native seminal RNase. while substitution of another portion (positions 76 and 80) attenuates the activity. Further, introduction of Gly at position 111 has been shown to increase the catalytic activity of RNase against double-stranded RNA. The variant and the wild-type (recombinant) protein were crystallized and their structures determined to a resolution of 2.0 Angstrom. Each of the mutated amino acids is seen in the electron density map. The main change observed in the mutant structure compared with the wild-type is the region encompassing residues 16-22, where the structure is more disordered. This loop is the region where the polypeptide chain of RNase A is cleaved by subtilisin to form RNase S, and undergoes conformational change to allow residues 1-20 of the RNase to swap between subunits in the covalent seminal RNase dimer.

An alternative to the origins of life theories: Amino acid-like DNA molecules with catalytic activity
Ang, DN; Suh, B; Westermann-Clark, E; Battersby, T; Benner, SA
FASEB J.13(7) (1999)

Synthesis of 2 '-deoxyisoguanosine 5 '-triphosphate and 2 '-deoxy-5-methylisocytidine 5 '-triphosphate
Jurczyk, SC; Kodra, JT; Park, JH; Benner, SA; Battersby, TR
Helv. Chim. Acta82(7) 1005-1015 (1999)
<Abstract>

The syntheses of the 5'-triphosphates of 2'-deoxyisoguanosine (= p(3)isoG(d)) and 2'-deoxy-5-methylisocytidine (= p(3)me(5)isoC(d)), two new bases for the genetic alphabet, are described. The triphosphates were synthesized from the corresponding nucleosides using a transient-protection procedure. The introduction of a methyl group at the 5-position of 2'-deoxyisocytidine remarkably improved the stability of the triphosphate. Characterization of the triphosphates included enzymatic incorporation opposite the complementary base in a template oligonucleotide.

A mild and efficient method for the preparation of 5 '-dimethoxytrityl-2 '-deoxynucleoside using poly(4-vinylpyridine)-costyrene
Karalkar, NB; Akerkar, VG; Salunkhe, MM
Indian J. Chem., Sect B38(3) 370-371 (1999)
<Abstract>

5'-O-4,4'-Dimethoxytrityl-2'-deoxynucleosides have been synthesized in high yield by the reaction of 2'-deoxynucleosides with 4, 4'-dimethoxytrityl chloride in acetonitrile using poly (4-vinylpyridine)-costyrene (styrene 10%).

Quantitative analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog
Battersby, TR; Ang, DN; Burgstaller, P; Jurczyk, SC; Bowser, MT; Buchanan, DD; Kennedy, RT; Benner, SA
J. Am. Chem. Soc.121(42) 9781-9789 (1999)
<Abstract>

5-(3 "-Aminopropynyl)-2'-deoxyuridine (dJ), a modi fled nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library;has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.

Catalysts, anticatalysts, and receptors for unactivated phosphate diesters in water
Zepik, HH; Benner, SA
J. Org. Chem.64(22) 8080-8083 (1999)
<Abstract>

A set of substituted bisguanidines have been prepared and examined for their ability to bind and catalyze the hydrolysis of uridylyl-3',5'-uridine (UpU), an unactivated RNA substrate in water. The unexpected result is that this set includes both catalysts (binding the transition state better than the ground state) and anticatalysts (binding the ground state better than the transition state), each with respectable rate enhancements and/or affinities, despite the fact that these molecules all have very similar structures. These results therefore show the level of sophistication that must be achieved in the conformational theory of small molecules if we hope to truly "design" supramolecular structures that bind preferentially to a transition state over the ground state.

Biological chemistry of copper-zinc superoxide dismutase and its link to amyotrophic lateral sclerosis
Lyons, TJ; Gralla, EB; Valentine, JS
Met. Ions Biol. Syst.36(1) 125-177 (1999)

An in vitro screening technique for DNA polymerases that can incorporate modified nucleotides. Pseudothymidine as a substrate for thermostable polymerases
Lutz, S; Burgstaller, P; Benner, SA
Nucl. Acids Res.27(13) 2792-2798 (1999)
<Abstract>

DNA polymerases are desired that incorporate modified nucleotides into DNA with diminished pausing, premature termination and infidelity. Reported here is a simple in vitro assay to screen for DNA polymerases that accept modified nucleotides based on a set of primer extension reactions, In combination with the scintillation proximity assay (SPA(TM)), this allows rapid and simple screening of enzymes for their ability to elongate oligonucleotides in the presence of unnatural nucleotides, A proof of the concept is obtained using pseudo-thymidine (psi T), the C-nucleoside analog of thymidine, as the unnatural substrate. The conformational properties of psi T arising from the carbon-carbon bond between the sugar and the base make it an interesting probe for the importance of conformational restraints in the active site of polymerases during primer elongation, From a pool of commercially available thermostable polymerases, the assay identified Taq DNA polymerase as the most suitable enzyme for the PCR amplification of oligonucleotides containing psi T. Subsequent experiments analyzing PCR performance and fidelity of Taq DNA polymerase acting on psi T are presented. This is the first time that PCR has been performed with a C-nucleoside.

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases
Lutz, MJ; Will, DW; Breipohl, G; Benner, SA; Uhlmann, E
Nucl. Nucl.18(3) 393-401 (1999)
<Abstract>

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coup led to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.

Synthesis and antiviral activities of 1,3-oxathiolanyl nucleosides with 5-hydroxymethyl substituent
Chun, MW; Choi, SP; Kim, MJ; Bae, CJ
Nucl. Nucl.18 615-616 (1999)
<Abstract>

Novel 1,3-oxathiolanyl pyrimidine nucleosides with 5-hydroxymethyl substituent were synthesized starting from D-mannose and evaluated for antiviral activities against HIV-1, HSV type 1,2 and HCMV.

The molecular origins of life - Assembling pieces of the puzzle
Benner, SA
Science283(5410) 2026-2026 (1999)

Chance and Necessity in Biomolecular Chemistry - Is Life as We Know It Universal?
Benner, SA; Switzer, CY
Simplicity and Complexity in Proteins and Nucleic Acids, ed. H. Frauenfelder, J. Deisenhofer, P.G. Wolynes, Dahlem University Press 339-363 (1999)

How small can a microorganism be?
Benner, SA
Size Limits of Very Small Microorganisms: Proceedings of a Workshop, Steering Group on Astrobiology of the Space Studies Board, National Research Council 126-135 (1999)

Post-genomic science: Converting primary structure into physiological function
Benner, SA; Trabesinger, N; Schreiber, D
Adv. Enz. Reg.38(1) 155-180 (1998)

A unified model of c-erbB receptor homo- and heterodimerisation
Chamberlin, SG; Davies, DE
Biochim. Biophys. Acta1384(2) 223-232 (1998)
<Abstract>

The c-erbB receptor tyrosine kinase family plays an important role in cell regulation. Receptor activation proceeds by the formation of receptor homo- and/or hetero-dimers and is promoted by the binding of a cognate ligand at the cell surface. While some experimental work has demonstrated that the formation of heterodimers can influence a cellular response, the extent of heterodimerisation has not been accurately assessed: the assortment of receptors and ligands gives rise to a complex combinatorial system for which intuitive prediction of homo- and hetero-dimerisation is difficult. We present a mathematical model which combines observations for homo-dimerisation with the additional interactions arising from the presence of multiple c-erbB receptors. We provide a simple explanation for the apparently conflicting results for binding studies carried out with either solubilised receptors, vesicles or cells and our model predicts binding behaviour which is compatible with published experimental findings for cells expressing either one or two c-erbB receptors. This model establishes the basis for interpretation of ligand binding experiments, where variations in the apparent ligand affinity can be attributed to changes in receptor expression or ligand preferences according to the binding profile. (C) 1998 Elsevier Science B.V. All rights reserved.

Structure prediction in a post-genomic environment: A secondary and tertiary structural model for the initiation factor 5A family
Gerloff, DL; Joachimiak, M; Cohen, FE; Cannarozzi, GM; Chamberlin, SG; Benner, SA
Biochem. Biophys. Res. Comm.251(1) 173-181 (1998)
<Abstract>

Two predictions have been prepared for the fold of initiation factor 5A (IF5A) starting from a set of homologous sequences. In the first, a secondary structural model was predicted for the protein in 1994, when only eleven homologs land no eubacterial homologs) had been sequenced. The second was made recently, after genome projects had generated a total of 33 sequences for the protein family from species of all three kingdoms of life. With the second set of sequences, but not with the first, it was possible to predict that the N-terminal domain of the protein folds in a possibly open beta-barrel/sandwich core structure, with a short helix capping one side of the barrel. We place the pair; of predictions in the public domain before an experimental structure is known. This example illustrates the impact of genome sequencing projects on structure prediction from sequence alignments. (C) 1998 Academic Press.

Origin of dimeric structure in the ribonuclease superfamily
Ciglic, MI; Jackson, PJ; Raillard, SA; Haugg, M; Jermann, TM; Opitz, JG; Trabesinger-Ruf, N; Benner, SA
Biochemistry37(12), ACS 4008-4022 (1998)
<Abstract>

To enable application of postgenomic evolutionary approaches to understand the divergence of behavior and function in ribonucleases (RNases), the impact of divergent sequence on the divergence of tertiary and quaternary structure is analyzed in bovine pancreatic and seminal ribonucleases, which differ by 23 amino acids, In a crystal, seminal RNase is a homodimer joined by two "antiparallel" intersubunit disulfide bonds between Cys-31 from one subunit and Cys-32' from the other and having composite active sites arising from the "swap" of residues 1-20 from each subunit. Specialized Edman degradation techniques have completed the structural characterization of the dimer hi solution, new crosslinking methods have been developed to assess the swap, and sequence determinants of quaternary structure have been explored by protein engineering using the reconstructed evolutionary history of the protein family as a guide. A single Cys at either position 32 (the first to be introduced during the divergent evolution of the family) or 31 converts monomeric RNase A into a dimer. Even with an additional Phe at position 31, another residue introduced early in the seminal lineage, swap is minimal, A hydrophobic contact formed by Leu-28, however, also introduced early in the seminal lineage, increases the amount of "antiparallel" connectivity of the two subunits and facilitates swapping of residues 1-20. Efficient swapping requires addition of a Pro at position 19, a residue also introduced early in the divergent evolution of the seminal RNase gene. Additional cysteines required for dimer formation are found to slow refolding of the protein through formation of incorrect disulfide bonds, suggesting a paradox in the biosynthesis of the protein. Further studies showed that the dimeric form of seminal RNase known in the crystal is not the only form in vivo, where a substantial amount of heterodimer is known, These data complete the acquisition of the background needed to understand the evolution of new structure, behavior, and function in the seminal RNase family of proteins.

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA
Opitz, JG; Ciglic, MI; Haugg, M; Trautwein-Fritz, K; Raillard, SA; Jermann, TM; Benner, SA
Biochemistry37(12), ACS 4023-4033 (1998)
<Abstract>

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Structure-function studies of ligand-induced epidermal growth factor receptor dimerization
Neelam, B; Richter, A; Chamberlin, SG; Puddicombe, SM; Wood, L; Murray, MB; Nandagopal, K; Niyogi, SK; Davies, DE
Biochemistry37(14), ACS 4884-4891 (1998)
<Abstract>

We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR(1-84)), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 mu M. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGF alpha(44-50) and the EGF point mutant L47A) and a C-terminally extended form of AR (AR(1-90)) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGF alpha(44-50) > L47A = L15A much greater than AR(1-90) > AR(1-84). Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR.

Recognition of a non-standard base pair by thermostable DNA polymerases
Lutz, MJ; Horlacher, J; Benner, SA
Bioorg. Med. Chem. Lett.8(10) 1149-1152 (1998)
<Abstract>

Examination of several commercially available thermostable DNA polymerases identifies 9 degrees N DNA polymerase as single enzyme that could incorporate two components of an expanded genetic alphabet, 2,4-diaminopyrimidine and xanthosine as deoxynucleoside triphosphate opposite their cognate base in a DNA template. (C) 1998 Elsevier Science Ltd. All rights reserved.

Recognition of 2 '-deoxyisoguanosine triphosphate by HIV-1 reverse transcriptase and mammalian cellular DNA polymerases
Lutz, MJ; Horlacher, J; Benner, SA
Bioorg. Med. Chem. Lett.8(5) 499-504 (1998)
<Abstract>

HIV-1 reverse transcriptase (RT) incorporates 2'-deoxyisoguanosine triphosphate (d-isoGTP) opposite thymidine (T) in a DNA template and opposite uracil (U) in an RNA template about 10 times more efficiently than the eukaryotic DNA polymerase a, both in the absence and presence of dATP. (C) 1998 Elsevier Science Ltd. All rights reserved.

The dark side of dioxygen biochemistry
Valentine, JS; Wertz, DL; Lyons, TJ; Liou, LL; Goto, JJ; Gralla, EB
Curr. Op. Chem Biol.2(2) 253-262 (1998)
<Abstract>

The cellular biochemistry of dioxygen is Janus-faced. The good side includes numerous enzyme-catalyzed reactions of dioxygen that occur in respiration and normal metabolism, while the dark side encompasses deleterious reactions of species derived from dioxygen that lead to damage of cellular components. These reactive oxygen species have historically been perceived almost exclusively as agents of the dark side, but it has recently become clear that they play beneficial roles as well.

Determination of solution structures of paramagnetic proteins by NMR
Turner, DL; Brennan, L; Chamberlin, SG; Louro, RO; Xavier, AV
Euro. Biophys. J. Biophys. Lett.27(4) 367-375 (1998)
<Abstract>

Standard procedures for using nuclear Overhauser enhancements (NOE) between protons to generate structures for diamagnetic proteins in solution from NMR data may be supplemented by using dipolar shifts if the protein is paramagnetic. This is advantageous since the electron-nuclear dipolar coupling provides relatively long-range geometric information with respect to the paramagnetic centre which complements the short-range distance constraints from NOEs. Several different strategies have been developed to date, but none of these attempts to combine data from NOEs and dipolar shifts in the initial stages of structure calculation or to determine three dimensional protein structures together with their magnetic properties. This work shows that the magnetic and atomic structures are highly correlated and that it is important to have additional constraints both to provide starting parameters for the magnetic properties and to improve the definition of the best fit. Useful parameters can be obtained for haem proteins from Fermi contact shifts; this approach is compared with a new method based on the analysis of dipolar shifts in haem methyl groups with respect to data from horse and tuna ferricytochromes c. The methods developed for using data from NOEs and dipolar shifts have been incorporated in a new computer program, PARADYANA, which is demonstrated in application to a model data set for the sequence of the haem octapeptide known as microperoxidase-8.

A combinatorial distance-constraint approach to predicting protein tertiary models from known secondary structure
Chelvanayagam, G; Knecht, L; Jenny, T; Benner, SA; Gonnet, GH
Folding Des.3(3) 149-160 (1998)
<Abstract>

Background: Distance geometry methods allow protein structures to be constructed using a large number of distance constraints, which can be elucidated by experimental techniques such as NMR. New methods for gleaning tertiary structural information from multiple sequence alignments make it possible for distance constraints to be predicted from sequence information alone. The basic distance geometry method can thus be applied using these empirically derived distance constraints. Such an approach, which incorporates a novel combinatoric procedure, is reported here. Results: Given the correct sheet topology and disulfide formations, the fully automated procedure is generally able to construct native-like C alpha models for eight small beta-protein structures. When the sheet topology was unknown but disulfide connectivities were included, ail sheet topologies were explored by the combinatorial procedure. Using a simple geometric evaluation scheme, models with the correct sheet topology were ranked first in four of the eight example cases, second in three examples and third in one example. if neither the sheet topology nor the disulfide connectivities were given a priori, all combinations of sheet topologies and disulfides were explored by the combinatorial procedure. The evaluation scheme ranked the correct topology within the top five folds for half the example cases. Conclusions: The combinatorial procedure is a useful technique for identifying a limited number of low-resolution candidate folds for small, disulfide-rich, beta-protein structures. Better results are obtained, however, if correct disulfide connectivities are known in advance, Combinatorial distance constraints can be applied whenever there are a sufficiently small number of finite connectivities. (C) Current Biology Ltd ISSN 1359-0278.

Synthesis of oligonucleotides containing 2 '-deoxyisoguanosine and 2 '-deoxy-5-methylisocytidine using phosphoramidite chemistry
Jurczyk, SC; Kodra, JT; Rozzell, JD; Benner, SA; Battersby, TR
Helv. Chim. Acta81(5) 793-811 (1998)
<Abstract>

The synthesis of oligonucleotides containing 2'-deoxy-5-methylisocylidine and 2'-deoxyisoguanosine using phosphoramidite chemistry in solid-phase oligonucleotide synthesis is described. Supporting previous observations, the N,N-diisobutylformamidine moiety was found to be a far superior protecting group than N-benzoyl for 2'-deoxy-5-methylisoeylidine. 2'-Deoxy-N-2-[(diisobutylamino)methylidene-5'(4,4'-dimethoxytrityl)-5-me thylisocytidine 3'-(2-cyanoethyl diisopropylphosphoramidite) (Ic) incorporated multiple consecutive residues during a standard automated synthesis protocol with a coupling efficiency >99% according to dimethoxytrityl release. Extending coupling times of the standard protocol to greater than or equal to 600 s using 2'-deoxy-N-6-[(diisobutylamino)methylidene]-5'-O-(dimethoxytrityl)-O-2-( diphenylcarbamoyl)isoguanosine, 3'-(2-cyanoethyl diisopropylphosphoramidite) (7e) led to successful incorporation of multiple consecutive 2'-deoxyisoguanosine bases with a coupling efficiency > 97% according to dimethoxytrityl release.

Synthesis of sulfoxides by the oxidation of sulfides with polymer supported periodate ion
Karalkar, NB; Salunkhe, MM; Talekar, KP; Maldar, NN
Indian J. Chem., Sect B37(11) 1184-1185 (1998)
<Abstract>

Aromatic and aliphatic sulfides have been oxidized by polymer-supported periodate ion to give sulfoxides in high yield and purity without undesirable side reaction.

Distorting duplex DNA by dimethylenesulfone substitution: A new class of "transition state analog" inhibitors for restriction enzymes
Blattler, MO; Wenz, C; Pingoud, A; Benner, SA
J. Am. Chem. Soc.120(11) 2674-2675 (1998)

Metal ion reconstitution studies of yeast copper-zinc superoxide dismutase: the "phantom" subunit and the possible role of Lys7p
Lyons, TJ; Nersissian, A; Goto, JJ; Zhu, H; Gralla, EB; Valentine, JS
J. Biol. Inorg. Chem.3(6) 650-662 (1998)
<Abstract>

Using a corrected molar extinction coefficient for yeast apo copper-zinc superoxide dismutase (CuZnSOD), we have confirmed that the metal binding properties of this protein in vitro differ greatly from those of the bovine and human CuZnSOD enzymes. Thus yeast apo CuZnSOD was found to bind only one Co2+ per protein dimer under the conditions in which the bovine and human CuZnSOD apoenzymes readily bind two per dimer. The spectroscopic properties characteristic of the two Cu2+ plus two Co2+ per dimer or four Cu2+ per dimer metal-substituted bovine apo CuZnSOD derivatives were obtained for the yeast apoprotein but by the addition of only half of the appropriate metals, i.e., one Cu2+ plus one Co2+ per dimer or two Cu2+ per dimer. This half-metallated yeast CuZnSOD has been characterized by UV-visible and EPR spectroscopy as well as by native polyacrylamide gel electrophoresis. We conclude that yeast apo CuZnSOD, unlike the bovine and human apoproteins, cannot be reconstituted fully with metal ions under the same conditions. Instead, only one subunit of the homodimer, the "normal" subunit, can be remetalled in a fashion reminiscent of the well-characterized bovine protein. The other "phantom" subunit is not competent to bind metals in this fashion. Furthermore, we have shown that CuZnSOD protein isolated from Saccharomyces cerevisiae that lacks the gene coding for the copper chaperone, Lys7p, contains only one metal ion, Zn2+, per protein dimer. The possibility that yeast CuZnSOD can exist in multiple conformational states may represent an increased propensity of the yeast protein to undergo changes that can occur in all CuZnSODs, and may have implications for amyotrophic lateral sclerosis.

Synthesis and biodistribution of a short nonionic oligonucleotide analogue in mouse with a potential to mimic peptides
Eschgfaller, B; Konig, M; Boess, F; Boelsterli, UA; Benner, SA
J. Med. Chem.41(3) 276-283 (1998)
<Abstract>

A nonionic RNA analogue of the sequence r(U(SO2)G(SO2)A(SO2)C) has been synthesized where each bridging phosphate diester is replaced by a dimethylene sulfone unit (rSNA). The rSNA was synthesized in solution from 3',5'-bishomo-beta-ribonucleoside derivatives as building blocks. Full experimemtal procedures are provided, and the product and all synthetic inter mediates are fully characterized. The tetramer is nonionic but highly dipolar due to multiple hydrogen bonding opportunities. It is freely soluble in water only at higher pH's, permitting it to be radiolabeled by exchange of the acidic protons cr. to the sulfones with tritiated water. The tritiated molecule was administered intravenously into the tail vein (2.6 mg/kg) of mice, and its distribution was monitored over 48 h. The rSNA, was widely distributed in the biological tissues, including the brain, and excreted in both the feces and the urine. The accumulation of radioactivity was significantly higher in liver and kidney than in other tissues. Radiolabel was recovered from the urine, analyzed by HPLC, and shown to be intact oligonucleotide sulfone. This is the first bioavailability study on a short nonionic oligonucleotide analogue, a class of molecules with potential biomedical applications.

Functionalization of nucleoside analogs: Regiospecific conversion of 4 ''-hydroxyl groups to thioesters and deoxygenation of 3 '-hydroxyl groups
Huang, Z; Benner, SA
Nuc. Nuc. Nuc. acids17(5), Taylor & Francis Group 895-899 (1998)
<Abstract>

Nucleoside analogs 3a-e were conveniently functionalized by regiospecifically converting 4 "-hydroxyl groups to thioesters 4a-e (>90% yield) and reducing 3'-hydroxyl groups to give building blocks 2a-e in 85-90% yields.

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion
Derkatch, IL; Bradley, ME; Liebman, SW
Proc. Natl. Acad. Sci. USA95(5) 2400-2405 (1998)
<Abstract>

[PSI+], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI+]. However, another non-Mendelian determinant, [PIN+], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI+] by Sup35p overproduction in [PIN+] strains, but has no effect on the propagation of [PSI+] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI+] strains. We argue that excess Sup45p inhibits [PSI+] seed formation. Because Sup35p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.

Redesigning nucleic acids
Benner, SA; Battersby, TR; Eschgfaller, B; Hutter, D; Kodra, JT; Lutz, S; Arslan, T; Baschlin, DK; Blattler, M; Egli, M; Hammer, C; Held, HA; Horlacher, J; Huang, Z; Hyrup, B; Jenny, TF; Jurczyk, SC; Konig, M; von Krosigk, U; Lutz, MJ; MacPherson, LJ; Moro
Pure Appl. Chem.70(2) 263-266 (1998)
<Abstract>

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.

Libraries for receptor-assisted combinatorial synthesis (RACS). The olefin metathesis reaction
Giger, T; Wigger, M; Audetat, S; Benner, SA
SynLett(6) 688 (1998)
<Abstract>

A library of alkenes is generated using the olefin metathesis reaction, and converted to a set of diols suitable for a receptor assisted combinatorial synthesis (RACS) experiment with borate as a linker.

Synthesis and properties of new chelating resin with a spacer containing alpha-nitroso-beta-naphthol as the functional group
Akerkar, VG; Karalkar, NB; Sharma, RK; Salunkhe, MM
Talanta46(6) 1461-1467 (1998)
<Abstract>

A new chelating ion-exchange resin with a spacer CH2-NH-C6H4- based on a microreticular chloromethylated styrene-divinylbenzene copolymer containing alpha-nitroso-beta-naphthol as a functional group has been synthesized. The sorption characteristics for manganese(II), iron(III), cobalt(II), nickel(II), copper(II), and zinc(II) have been investigated over the pH range 1.0-7.0. The resin is highly stable in acidic and alkaline medium. Iron(III) and cobalt(II); copper(II) and iron(III) are separated very effectively in a column operation by stepwise elution. (C) 1998 Elsevier Science B.V. All rights reserved.

Bona fide predictions of protein secondary structure using transparent analyses of multiple sequence alignments
Benner, SA; Cannarozzi, G; Gerloff, D; Turcotte, M; Chelvanayagam, G
Chem. Rev.97(8) 2725-2843 (1997)

Redesigning nucleic acids
Benner, SA
FASEB J.11(9) (1997)

Genetic and environmental factors affecting the de novo appearance of the [PSI+] prion in Saccharomyces cerevisiae
Derkatch, IL; Bradley, ME; Zhou, P; Chernoff, YO; Liebman, SW
Genetics147(2) 507-519 (1997)
<Abstract>

It has previously been shown that yeast prion [PSI+] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI+] and [psi(-)] yeast strains results in two classes of [psi(-)] derivatives: Pin(+), in which [PSI+] can be reinduced by Sup35p overproduction, and Pin(-), in which overexpression of the complete SUP35 gene does not lead to the [PSI+] appearance. However, in both Pin(+) and Pin(-) derivatives [PSI+] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI+] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin(+) [psi(-)] derivatives are observed in Pin(-) [psi(-)] derivatives. Genetic analyses show that the Pin(+) phenotype is determined by a non-Mendelian factor, which, unlike the [PSI+] prion, is independent of the Sup35p N-terminal domain. A Pin(-) [psi(-)] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI+] curing by Hsp104 overproduction resulted exclusively in Pin(+) [psi(-)] derivatives. We hypothesize that in addition to the [PSI+] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin(+) phenotype.

Recognition of uncharged polyamide-linked nucleic acid analogs by DNA polymerases and reverse transcriptases
Lutz, MJ; Benner, SA; Hein, S; Breipohl, G; Uhlmann, E
J. Am. Chem. Soc.119(13) 3177-3178 (1997)

Targeting determinants and proposed evolutionary basis for the sec and the delta pH protein transport systems in chloroplast thylakoid membranes
Henry, R; Carrigan, M; McCaffery, M; Ma, XY; Cline, K
J. Cell. Biol.136(4) 823-832 (1997)
<Abstract>

Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems, Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity, The results showed that: (n) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system, A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway, This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.

The B-12-dependent ribonucleotide reductase from the archaebacterium Thermoplasma acidophila: An evolutionary solution to the ribonucleotide reductase conundrum
Tauer, A; Benner, SA
Proc. Natl. Acad. Sci. USA94(1) 53-58 (1997)
<Abstract>

A coenzyme B-12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced, Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli, The deduced amino acid sequence shows that the catalytic domain of the B-12-dependent enzyme from T, acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man, The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not, Evolutionary bridges created by the T. acidophila sequence and that of a B-12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B-12-dependent enzyme as well, These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme, Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B-12 nor Fe cofactors, A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B-12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases, A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in ''post-genomic'' biochemistry.

A predicted consensus structure for the C-terminus of the beta and gamma chains of fibrinogen
Gerloff, DL; Cohen, FE; Benner, SA
Proteins27(2) 279-289 (1997)
<Abstract>

A secondary structure has been predicted for the C termini of the fibrinogen beta and gamma chains from an aligned set of homologous protein sequences using a transparent method that extracts conformational information from patters of variation and conservation, parsing strings, and patterns of amphiphilicity. The structure is modeled to form two domains, the first having a core parallel sheet flanked on one side by at least two helices and on the other by an antiparallel amphiphilic sheet, with an additional helix connecting the two sheets. The second domain is built entirely from beta strands. (C) 1997 Wiley-Liss, Inc.

A predicted consensus structure for the N-terminal fragment of the heat shock protein HSP90 family
Gerloff, DL; Cohen, FE; Korostensky, C; Turcotte, M; Gonnet, GH; Benner, SA
Proteins27(3) 450-458 (1997)
<Abstract>

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment). (C) 1997 Wiley-Liss, Inc.

An analysis of simultaneous variation in protein structures
Chelvanayagam, G; Eggenschwiler, A; Knecht, L; Gonnet, GH; Benner, SA
Prot. Eng.10(4) 307-316 (1997)
<Abstract>

The simultaneous substitution of pairs of buried amino acid side chains during divergent evolution has been examined in a set of protein families with known crystal structures, A weak signal is found that shows that amino acid pairs near in space in the folded structure preferentially undergo substitution in a compensatory way, Three different physicochemical types of covariation 'signals' were then examined separately, with consideration given to the evolutionary distance at which different types of compensation occur. Where the compensatory covariation tends towards retaining the combined residue volumes, the signal is significant only at very low evolutionary distances. Where the covariation compensates for changes in the hydrogen bonding, the signal is strongest at intermediate evolutionary distances. Covariations that compensate for charge variations appeared with equal strength at all the evolutionary distances examined, A recipe is suggested for using the weak covariation signal to assemble the predicted secondary structural elements, where the evolutionary distance, covariation type and weighting are considered together with the tertiary structural context (interior or surface) of the residues being examined.

Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry
Wigger, M; Nawrocki, JP; Watson, CH; Eyler, JR; Benner, SA
Rapid Comm. Mass Spec.11(16) 1749-1752 (1997)
<Abstract>

A model experiment for the 'on-line' screening of substrate libraries by enzymes using combinatorial Libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed, The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and components of a H-gamma-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored, It shows the feasibility of two-dimensional screening of substrate libraries by ESI-FTICR mass spectrometry. (C) 1997 John Wiley & Sons, Ltd.

Synthesis of an N-alkyl derivative of 2'-deoxyisoguanosine
Kodra, JT; Benner, SA
SynLett(8) 939-2843 (1997)
<Abstract>

3',5'-O-bis-tert-butyldimethylsilyl-2'deoxyguanosine is converted in two steps to 3',5'-O-tert-butyldimethylsilyl-6-O-aryl2'-deoxyxanthosine. This compound is used to make a 2'-deoxyisoguanosine analog with a functionalized side chain.

Developing new synthetic catalysts. How nature does it
Benner, SA; Jermann, TM; Opitz, JG; Raillard, SA; Zankel, TR; Trautwein-Fritz, K; Stackhouse, J; Ciglic, MI; Haugg, M; Trabesinger-Ruf, N; Weinhold, EG
Acta Chem. Scand.50(3) 243-248 (1996)
<Abstract>

Paleomolecular biochemistry is a new field of science that seeks to understand how life emerged and developed in interaction with its geophysical surroundings. It is an experimental science, involving reconstruction of extinct biomolecules in the laboratory, studying their properties in the laboratory, and inferring details of their behavior and function in the context of geological data. An outline is provided of some tools of this field, together with its application to the study of two specific systems, ribonuclease and alcohol dehydrogenase.

Targeting the epidermal growth factor receptor for therapy of carcinomas
Davies, DE; Chamberlin, SG
Biochem. Pharmacol.51(9) 1101-1110 (1996)
<Abstract>

As a group, the carcinomas represent a substantial proportion of all human malignancies, but, with relatively few exceptions, current treatments are ineffective. Modification of existing chemotherapeutic agents has not led to significant improvements in the survival of carcinoma patients, and development of new therapeutic strategies is imperative. It is now becoming apparent that activation of the epidermal growth factor receptor (EGF-R) has much wider implications than a straightforward stimulation of cell division. The pleiotropic effects of EGF-R signalling may influence tumour behaviour and the response of carcinomas to treatment; these are important considerations for the development of new therapies that aim to exploit the expression or modulate the function of the EGF-R in these tumours.

The interaction of a chimeric EGF containing the C-tail of TGF alpha with the EGF receptor reveals hidden ligand complexities.
Puddicombe, S; Wood, L; Chamberlin, S; Davies, D
FASEB J.10(6) 2171-2171 (1996)

The mitogenic effect of low affinity ligands is enhanced by overexpression of EGF receptors
Davies, D; Adam, R; Murray, M; Niyogi, S; Chamberlin, S
FASEB J.10(6) 2172-2172 (1996)

Amphiregulin is a poor effector of ligand-induced EGF receptor dimerisation.
Richter, A; Neelam, B; Chamberlin, S; Davies, D
FASEB J.10(6) 2173-2173 (1996)

Catalysis: The omnipresent phenomenon.
Benner, SA
FASEB J.10(6) (1996)

A high intensity signal is required for EGF receptor mediated morphogenesis and migration.
Solic, N; Murray, M; Chamberlin, S; Niyogi, S; Davies, D
FASEB J.10(6) (1996)

A mathematical model of c-erbB receptor dimerisation.
Chamberlin, S; Davies, D
FASEB J.10(6) (1996)

Pseudogenes in ribonuclease evolution: A source of new biomacromolecular function?
TrabesingerRuef, N; Jermann, T; Zankel, T; Durrant, B; Frank, G; Benner, SA
FEBS Lett.382(3) 319-322 (1996)
<Abstract>

Bovine seminal ribonuclease (RNase) diverged from pancreatic RNase after a gene duplication ca. 35 million years ago. Members of the seminal RNase gene family evidently remained as unexpressed pseudogene for much of its evolutionary history. Between 5 and 10 million years ago, however, after the divergence of kudu but before the divergence of ox, evidence suggests that the pseudogene was repaired and expressed. Intriguingly, detailed analysis of the sequences suggests that the repair may have involved gene conversion, transfer of information from the pancreatic gene to the RNase pseudogene. Further, the ratio of non-silent to silent substitutions suggests that the pancreatic RNases are divergently evolving under functional constraints, the seminal RNase pseudogenes are diverging under no functional constraints, while the genes expressed in the seminal plasma are evolving extremely rapidly in their amino acid sequences, as if to fulfil a new physiological role.

Induction of anchorage-independent growth by amphiregulin
Adam, RM; Chamberlin, SG; Davies, DE
Growth Factors13 193-203 (1996)
<Abstract>

We have previously shown that the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AR) exhibits low potency as a result of its C-terminal truncation. This led us to investigate whether its inability to promote anchorage-independent growth (AIG) of normal cells arose because of its compromised interaction with EGFR. Wild type AR(1-84) was tested in AIG and mitogenesis assays using NRK-49F or NRG/HER fibroblasts. In contrast to NRG/HER cells, the response of NRK-49F fibroblasts to AR was much lower than expected. As the effect of AR was heparin-insensitive, contributions from heparan sulphate proteoglycan interactions could not explain the differing sensitivities of the cells. Comparison of the effects of AR on two additional cell lines indicated that low EGFR number correlated with AR insensitivity: this suggested that the low potency of AR precluded activation of sufficient receptors to elicit a response. Consistent with this proposal, a modified form of AR (AR(1-90(leu86))) with enhanced potency was able to induce AIG of NRK-49F fibroblasts. Thus, the ability of AR to promote AIG is determined both by ligand potency and the EGFR complement of cells.

Synthesis and characterization of non-standard nucleosides and nucleotides bearing the acceptor-donor-donor pyrimidine analog 6-amino-3-methylpyrazin-2(1H)-one
Voegel, JJ; Benner, SA
Helv. Chim. Acta79(7) 1863-1880 (1996)
<Abstract>

6-Aminopyrazin-2(1H)-one, when incorporated as a pyrimidine-base analog into an oligonucleotide chain, presents a H-bond acceptor-donor-donor pattern to a complementary purine analog. When paired with the corresponding donor-acceptor-acceptor purine in oligonucleotides, the heterocycle selectively contributes to the stability of the duplex, presumably by forming a base pair of Warson-Click geometry joined by a non-standard H-bonding pattern. Aspects of the nucleoside chemistry, including syntheses of the beta-furanosyl ribonucleoside 1, the ribonucleoside triphosphate 2 and the ribonucleoside bisphosphate 3 of 6-aminopyrazin-2(1H)-one are reported here. In aqueous solution, the ribonucleoside 1 was found to undergo acid- and base-catalyzed rearrangement with an apparent half-life of ca. 63 h at neutral pH and 30 degrees. The rearrangement appears to be specific acid- and base-catalyzed. The thermodynamically most stable compound formed during this rearrangement reaction was isolated by HPLC and shown to be the beta-pyranosyl form 4 of the 6-aminopyrazin-2(1H)-one nucleoside in its C-4(1) chair conformation. This reactivity of 1 under physiological conditions may explain why Nature does not use this particular heterocyclic system to implement an acceptor-donor-donor H-bonding pattern in the genetic alphabet.

Synthesis, molecular recognition, and enzymology of oligonucleotides containing the non-standard base pair between 5-aza-7-deazaisoguanine and 6-amino-3-methylpyrazin-2(1H)-one, a donor-acceptor-acceptor purine analog and an acceptor-donor-donor pyrimidine analog
Voegel, JJ; Benner, SA
Helv. Chim. Acta79(7) 1881-1898 (1996)
<Abstract>

A 6-aminopyrazin-2(1H)-one (pyADD), when incorporated as a pyrimidine-base analog into an oligonucleotide chain, presents a H-bond acceptor-donor-donor pattern to 5-aza-7-deazaisoguanine (puDAA), the complementary donor-acceptor-acceptor purine analog. Reported here are the syntheses of the phosphoramidite of the 2'-deoxyribonucleoside bearing the puDAA base, oligonucleotides containing this nucleoside unit, the enzyme-assisted synthesis of oligoribonucleotides containing the pyADD ribonucleoside, and the molecular-recognition properties of this non-standard base pair in an oligonucleotide context. A series of melting experiments suggests that the pyADD . puDAA base pair contributes to the relative stability of a duplex structure approximately the same as an A . T base pair, and significantly more than mismatches between these non-standard bases and certain standard nucleobases. The pyADD nucleoside bisphosphate is accepted by T4 RNA ligase, but the triphosphate of the pyADD nucleoside was not incorporated by T7 RNA polymerase opposite the puDAA nucleobase in a template. Oligonucleotides containing the pyADD base slowly undergo a reversible first-order reaction, presumably an epimerization process to give the alpha-D-anomer. These experiments provide the tools for laboratory-based use of the pyADD . puDAA base pair as a component of an oligonucleotide-like molecular-recognition system based on an expanded genetic alphabet.

Nonionic analogs of RNA with dimethylene sulfone bridges
Richert, C; Roughton, AL; Benner, SA
J. Am. Chem. Soc.118(19) 4518-4531 (1996)
<Abstract>

Analogs of RNA have been synthesized where each of the phosphodiester linking groups is replaced by dimethylene sulfone units (sulfone-linked nucleic acid analogs of RNA, or ''rSNAs''). These are the first fully nonionic analogs of RNA to be prepared as oligomers. Sequences leading to the octamer 5'-r(A(SO2)U(SO2)G(SO2)U(SO2)C(SO2)-A(SO2)U)-3' have been prepared from 3',5'-bishomo-beta-ribonucleoside derivatives as building blocks prepared from diacetone D-glucose, and their chemistry has been explored. Coupling was performed in solution via S(N)2 reactions between a thiol from one fragment and a bromide from the other, oxidation of the resulting thioether to the sulfone, and deprotection of a terminal primary hydroxyl group and regioselective conversion of it-in the presence of secondary hydroxyl groups--to an active group (thiol or bromide) to yield another fragment for coupling. Base-labile protecting groups were used for the nucleobases, and one-step full deprotection was achieved using 1 M NaOH. The target octamer and each isolated intermediate were characterized by NMR, UV spectroscopy, and mass spectrometry. While chemical reactions involving longer rSNAs were in several cases retarded relative to analogous reactions with monomers, some rates were enhanced. In water, the rSNA octamer displayed a thermal transition in the UV spectrum above 65 degrees C with a large hyperchromicity. The behaviors of rSNAs suggest roles for the polyanionic backbone in DNA and RNA beyond its role in conferring aqueous solubility. The repeating anionic charges in natural oligonucleotides evidently also control the potent molecular recognition properties of these richly functionalized molecules, direct strand-strand interactions to the part of the biopolymer distant from the backbone (the Watson-Crick edge of the nucleobases), cause the polymer to favor an extended conformation, and ensure that the physical properties of the oligonucleotide are largely independent of its sequence. This suggests structural features that must be built into nonionic oligonucleotide analogs generally.

The significance of valine 33 as a ligand-specific epitope of transforming growth factor alpha
Puddicombe, SM; Chamberlin, SG; MacGarvie, J; Richter, A; Drummond, DR; Collins, J; Wood, L; Davies, DE
J. Biol. Chem.271(26), ASBMB 15367-15372 (1996)
<Abstract>

Although binding of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) to the EGF receptor (EGFR) is mutually competitive, their binding is not identical, and their biological activities are not always equivalent, To probe for ligand-specific interactions, me have synthesized analogues of TGF alpha with modifications to the residue lying between the fourth and fifth cysteines (the ''hinge''). Although this residue Lies in a structurally conserved region of the protein, it is not conserved within the EGFR ligand family. Our results show that in TGF alpha there is a preference for a bulky hydrophobic hinge residue; this contrasts with EGF, for which a hydrogen bond donor functionality is preferred. Sequence analysis of the human EGFR Ligands revealed that the nature of the hinge residue correlated with the sequence in the B-loop beta-sheet. As this region is an important determinant in recognition of TGF alpha by the chicken EGFR, we assessed the mitogenicity of the TGF alpha hinge mutants, as well as the other EGFR ligands, using chicken embryo fibroblasts. The preference of the chicken EGFR for TGF alpha hinge mutants with hydrophobic side chains paralleled that of the human EGFR. Betacellulin and heparin-binding EGF-like growth factor also possess an hydrophobic hinge; both were at least as potent as TGF alpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGF alpha. We propose that EGFR ligands can be subclassified into TGF alpha-like and EGF-like and that this is of functional significance, identifying a potential mechanism whereby EGFR can discriminate between its ligands.

The interaction of an epidermal growth factor transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities
Puddicombe, SM; Wood, L; Chamberlin, SG; Davies, DE
J. Biol. Chem.271(48), ASBMB 30392-30397 (1996)
<Abstract>

It has been assumed that substitution of homologous regions of transforming growth factor alpha (TGF-alpha) into epidermal growth factor (EGF) can be used to probe ligand-receptor recognition without detrimental effects on ligand characteristics for the human EGF receptor (EGFR), We show that a chimera of murine (m) EGF in which the carboxyl-terminal tail is substituted for that of TGF-alpha (mEGF/TGF-alpha(44-50)) results in complex features that belie this initial simplistic assumption, Comparison of EGF and mEGF/TGF-alpha(44-50) in equilibrium binding assays showed that although the relative binding affinity of the chimera was reduced 80-200-fold, it was more potent than EGF in mitogenesis assays using NR6/HER cells, This superagonist activity could not be attributed to differences in ligand processing or to binding to other members of the c-erbB family. It appeared to be due, in part, to choice of an EGFR overexpressing target cell where high receptor number compensated for the low affinity of the ligand; it also appeared to be related to the ability of the chimera to activate the EGFR tyrosine kinase, Thus, when EGFR autophosphorylation was measured, mEGF/TGF-alpha(44-50) was more potent than EGF, despite its low affinity, When tested using chicken embryo fibroblasts, substitution of the TGF-alpha carboxyl-terminal tail into mEGF failed to enhance its binding affinity for chicken EGFRs; however, the chimera was intermediate in potency between TGF-alpha and mEGF in mitogenesis assays, Our results suggest a contextual requirement for EGFR recognition which is ligand-specific, Further, the unpredictable responses to chimeric ligands underline the complex nature of the processes of ligand recognition, receptor activation, and the ensuing cellular response.

Chimera of dimethylene sulfone-, methyl sulfide-, and methyl sulfoxide-linked ribonucleotides and DNA
Baeschlin, DK; Hyrup, B; Benner, SA; Richert, C
J. Org. Chem.61(21) 7620-7626 (1996)

Differential discrimination of DNA polymerases for variants of the non-standard nucleobase pair between xanthosine and 2,4-diaminopyrimidine, two components of an expanded genetic alphabet
Lutz, MJ; Held, HA; Hottiger, M; Hubscher, U; Benner, SA
Nucl. Acids Res.24(7) 1308-1313 (1996)
<Abstract>

Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) were examined for their ability to incorporate components of an expanded genetic alphabet in different forms. Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanthosine (c(7)dX), both able to adopt a hydrogen bonding acceptor-donor-acceptor pattern on a purine nucleus (puADA). Thus these heterocycles are able to form a non-standard nucleobase pair with 2,4-diaminopyrimidine (pyDAD) that fits the Watson-Crick geometry, but is joined by a non-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c(7)dX was significantly lower and also independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E.coli DNA polymerase I failed to incorporate d(pyDAD)TP opposite c(7)dX.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein
Lyons, TJ; Liu, HB; Goto, JJ; Nersissian, A; Roe, JA; Graden, JA; Cafe, C; Ellerby, LM; Bredesen, DE; Gralla, EB; Valentine, JS
Proc. Natl. Acad. Sci. USA93(22) 12240-12244 (1996)
<Abstract>

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the mild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.

Analysis of combinatorial libraries using electrospray Fourier transform ion cyclotron resonance mass spectrometry
Nawrocki, JP; Wigger, M; Watson, CH; Hayes, TW; Senko, MW; Benner, SA; Eyler, JR
Rapid Comm. Mass Spec.10(14) 1860-1864 (1996)
<Abstract>

Electrospray ionization coupled with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to provide information about complete combinatorial libraries of small peptides containing 10(3)-10(4) components, The fidelity of attempted synthesis steps can be ascertained rapidly, and, when the extremely high resolution FTICR mass spectra are combined with appropriate computer simulation, both diversity and degeneracy of the libraries as synthesized can be assessed.

Protein structure prediction
Benner, SA; Geroff, DL; Rozzell, JD
Science274(5292) 1448-1449 (1996)

Four step synthesis of a 5'-deoxy-5'-iodomethylthymidine
Baeschlin, DK; Daube, M; Blattler, MO; Benner, SA; Richert, C
Tet. Lett.37(10) 1591-1592 (1996)
<Abstract>

The conversion of an alkylsulfonate to an iodide with triphenylphosphine/iodine in benzene has been performed for a nucleoside. Starting from thymidine, 3'-protected 5'-deoxy-5'-iodomethylthymidine was synthesized in 4 steps.

Reconstructing ancient forms of life
Benner, SA
J. Cell. Biol.(19) 200-200 (1995)

MODULATION OF THE RECEPTOR-BINDING AFFINITY OF AMPHIREGULIN BY MODIFICATION OF ITS CARBOXYL-TERMINAL TAIL
Adam, R; Drummond, DR; Solic, N; Holt, SJ; Sharma, RP; Chamberlin, SG; Davies, DE
Biochim. Biophys. Acta1266(1) 83-90 (1995)
<Abstract>

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR(1-84)), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR(1-90)) and a similarly extended construct with a Met(86) to Leu substitution (AR1-90((leu86))) were expressed as recombinant proteins in yeast, purified by heparin affinity and C-18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu(86) > met(86) > wt. The AR1-90((leu86))) construct was found to be 50- to 100-fold more active than wild type AR(1-84), consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR(1-90), which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR(1-84). This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.

Immunosuppressive activity of angiogenin in comparison with bovine seminal ribonuclease and pancreatic ribonuclease
Matousek, J; Soucek, J; Riha, J; Zankel, TR; Benner, SA
Comp. Biochem. Phys. B112(2) 235-241 (1995)
<Abstract>

Angiogenin, a member of the pancreatic-like ribonuclease family with a special biological action (RISBAses), is a basic protein that induces blood vessel formation. Another member of these special ribonucleases, bovine seminal ribonuclease (BS RNase), displays biological properties, including aspermatogenic, embryotoxic, antitumor and immunosuppressive activities. The effects of two angiogenin preparations tested on the biological activities mentioned above are reported and compared with those of BS RNase and RNase A. In contrast to RNase A, which was ineffective in all biological activities tested, angiogenin suppressed significantly the proliferation of human lymphocytes stimulated by phytohemagglutinin or concanavalin A or by allogenic human lymphocytes (mixed lymphocyte culture). However, angiogenin did not affect the growth of human tumor cell lines, development of cow acid mouse embryos and spermatogenicity in mice. On the basis of these results, angiogenin is the first monomeric ribonuclease described so far that displays immunosuppressive activity similar to that of the dimeric BS RNase. The immunosuppressive activity of angiogenin might synergize with the effect on neovascularization of tumor tissues and thus contribute to the development of tumor.

pH-independent triple helix formation by an oligonucleotide containing a pyrazine donor-donor-acceptor base
Vonkrosigk, U; Benner, SA
J. Am. Chem. Soc.117(19) 5361-5362 (1995)

Crystal structure of a dimethylene sulfone-linked ribodinucleotide analog
Roughton, AL; Portmann, S; Benner, SA; Egli, M
J. Am. Chem. Soc.117(27) 7249-7250 (1995)

CONTRIBUTION OF THE TRANSFORMING GROWTH-FACTOR-ALPHA B-LOOP BETA-SHEET TO BINDING AND ACTIVATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR
Richter, A; Drummond, DR; Macgarvie, J; Puddicombe, SM; Chamberlin, SG; Davies, DE
J. Biol. Chem.270(4), ASBMB 1612-1616 (1995)
<Abstract>

We have exploited the differences in binding affinities of the chicken epidermal growth factor (EGF) receptor for EGF and transforming growth factor alpha (TGF alpha) to study the role of the B-loop beta-sheet of these ligands in receptor recognition and activation. Although EGF and TGF alpha share similar secondary and tertiary structures imposed by three highly conserved intramolecular disulfide bonds, they have only 30-40% overall sequence identity. The B-loop beta-sheet is the major structural element in EGF and TGF alpha, but sequence similarity in this region is low. To investigate its role in receptor binding, we constructed two chimeric growth factors (mEGF/hTGF alpha(21-40) and mEGF/hTGF alpha(21-32)) composed of the murine EGF (mEGF) amino acid sequence with residues 21-30 of the B-loop beta-sheet replaced by the equivalent residues of human TGF alpha (hTGF alpha); in chimera mEGF/hTGF alpha(21-32), asparagine 32, which lies at the boundary of the amino and carboxyl domains of mEGF, was also replaced by its hTGF alpha counterpart (valine). In initial studies using unpurified medium, it was found that the recombinant growth factors exhibited differing mitogenic potencies (mEGF/hTGF alpha(21-32) > mEGF/hTGF alpha(21-30) > mEGF) when assayed on chicken fibroblasts, even though they were equivalent in mitogenesis assays us ing cells expressing the human EGF receptor, After purification, mEGF/hTGF alpha(21-32) was found to be 50 times more potent than mEGF in the chick fibroblast mitogenesis assay and exhibited a 10-fold increase in relative affinity for the chicken EGF receptor; both growth factors still exhibited equivalent mitogenic and receptor binding activity when tested on cells expressing human EGF receptors. We conclude that the B-loop beta-sheet of hTGF alpha is an important determinant of EGF receptor binding affinity and biological activity.

CONSTRAINED PEPTIDE ANALOGS OF TRANSFORMING GROWTH-FACTOR-ALPHA RESIDUES CYSTEINE-21-32 ARE MITOGENICALLY ACTIVE - USE OF PROLINE MIMETICS TO ENHANCE BIOLOGICAL POTENCY
Chamberlin, SG; Sargood, KJ; Richter, A; Mellor, JM; Anderson, DW; Richards, NGJ; Turner, DL; Sharma, RP; Alexander, P; Davies, DE
J. Biol. Chem.270(36), ASBMB 21062-21067 (1995)
<Abstract>

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro(30) into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys(21)-Cys(32)). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC(50) values in the range 130-330 mu M; in the presence of low doses of TGF-alpha, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-alpha alone. These effects are consistent with the peptides acting via the same mechanism as TGF-alpha. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the beta-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.

X-ray crystal-structure of a dimethylene sulfone-bridged ribonucleotide dimer in a single-stranded state
Hyrup, B; Richert, C; Schulteherbruggen, T; Benner, SA; Egli, M
Nucl. Acids Res.23(13) 2427-2433 (1995)
<Abstract>

A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide, r(Aso(2)U), where a bridging non-ionic dimethylene sulfone linker replaces the phosphodiester linking group found in natural RNA, Crystals of the single-stranded state of r(Aso(2)U) were obtained from water at 50 degrees C, In these crystals, one hydrogen bond is formed between bases from different strands and base stacking occurs in intermolecular 'home-A' and 'homo-U' stacks, Similar to typical oligoribonucleotides, the ribose rings adopt N-type conformations and dihedral angles chi are in the anti range, The all-trans rotamer of the CH2-SO2-CH2-CH2 bridge was found, which leads to a large adenine-uracil distance, Qualitative analysis of a NOESY spectrum of the Aso(2)U part in r(Uso(2)Cso(2)Aso(2)U) dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation observed in the crystal is also populated in solution, Comparison with the structure of r(Gso(2)C), which has been crystallized in the Watson-Crick paired state, shows that a rotation around zeta by +112 degrees leads from the observed, single-stranded state to a conformation that is compatible with formation of a duplex, A concerted translgauche flip of alpha and gamma then yields the standard conformer of A-type RNA helices, From the observed structure of r(Gso(2)C) and other oligonucleotides it is anticipated that this flip will also revert the ribose pucker from C2'-exo to C3'-endo.

Reconstructing the evolutionary history of the artiodactyl ribonuclease superfamily
Jermann, TM; Opitz, JG; Stackhouse, J; Benner, SA
Nature374(6517) 57-59 (1995)
<Abstract>

THE sequences of proteins from ancient organisms can be reconstructed from the sequences of their descendants by a procedure that assumes that the descendant proteins arose from the extinct ancestor by the smallest number of independent evolutionary events ('parsimony')(1,2). Tbe reconstructed sequences can then be prepared in the laboratory and studied(3,4). Thirteen ancient ribonucleases (RNases) have been reconstructed as intermediates in the evolution of the RNase protein family in artiodactyls (the mammal order that includes pig, camel, deer, sheep and ox)(5). The properties of the reconstructed proteins suggest that parsimony yields plausible ancient sequences. Going back in time, a significant change in behaviour, namely a fivefold increase in catalytic activity against double-stranded RNA, appears in the RNase reconstructed for the founding ancestor of the artiodactyl lineage, which lived about 40 million years ago(6). This corresponds to the period when ruminant digestion arose in the artiodactyls, suggests that contemporary artiodactyl digestive RNases arose from a non-digestive ancestor, and illustrates how evolutionary reconstructions can help in tbe understanding of physiological function within a protein family(7-9).

Uncertainty in ancient phylogenies - reply
Benner, SA; Jermann, TM; Opitz, JG; Stackhouse, J; Knecht, LJ; Gonnet, GH
Nature377(6545) 109-110 (1995)

Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns
Horlacher, J; Hottiger, M; Podust, VN; Hubscher, U; Benner, SA
Proc. Natl. Acad. Sci. USA92(14) 6329-6333 (1995)
<Abstract>

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N-1-methyloxoformycin B (pi) has been investigated, The kappa-X and kappa-pi base pairs are joined by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs, Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity, With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template, With d pi in the template, no incorporation of dKTP was observed with HIV reverse transcriptase, The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa, Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base, These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.

Predicted secondary and supersecondary structure for the serine-threonine-specific protein phosphatase family
Jenny, TF; Gerloff, DL; Cohen, MA; Benner, SA
Proteins21(1) 1-10 (1995)
<Abstract>

A bona fide consensus prediction for the secondary and supersecondary structure of the serine-threonine specific protein phosphatases is presented. The prediction includes assignments of active site segments, an internal helix, and a region of possible 3(10) helical structure. An experimental structure for a member of this family of proteins should appear shortly, allowing this prediction to be evaluated. (C) 1995 Wiley-Liss, Inc.

A consensus prediction of the secondary structure for the 6-phospho-β-D-galactosidase superfamily
Gerloff, DL; Benner, SA
Proteins21(4) 273-281 (1995)
<Abstract>

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-beta-D-galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (alpha-beta class), with the core beta strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel beta sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C + D) and (D + E), respectively, and an analogous beta-alpha unit (strand G and helix 7) toward the end of the sequence, The resemblance of the supersecondary model to the tertiary structure formed by 8-fold alpha-beta barrel proteins is almost certainly not coincidental. (C) 1995 Wiley-Liss, Inc.

A predicted consensus structure for the protein-kinase C2 homology (C2H) domain, the repeating unit of synaptotagmin
Gerloff, DL; Chelvanayagam, G; Benner, SA
Proteins22(4) 299-310 (1995)
<Abstract>

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred. (C) 1995 Wiley-Liss, Inc.

The phospho-β-galactosidase and synaptotagmin predictions
Benner, SA; Gerloff, D; Chelvanayagam, G
Proteins23(3) 446-453 (1995)
<Abstract>

Two bona fide consensus predictions of secondary and tertiary structure in a protein family, made and announced before experimental structures were known, are evaluated in light of the subsequently determined experimental structures. The first, for phospho-beta-galactosidase, identified the core strands of an 8-fold alpha-beta barrel, and identified the 8-fold alpha-beta barrel itself, which was found in the subsequently determined experimental structure to be the core folding domain. The second, for synaptotagmin, identified seven out of eight beta-strands in the structure correctly, missing only a noncore strand. Three preferred ''topologies'' were selected from several hundred thousand possible topologies of these seven predicted strands using a rule-based analysis. The subsequently determined experimental structure showed that these seven strands in synaptotagmin adopt one of the three preferred topologies. We were unable, however, to identify the correct topology from among these three topologies. (C) 1995 Wiley-Liss, Inc.

Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity
Weinhold, EG; Benner, SA
Prot. Eng.8(5) 457-461 (1995)
<Abstract>

Analysis of a crystal structure of alcohol dehydrogenase (Adh) from horse liver suggests that Trp54 in the homologous yeast alcohol dehydrogenase prevents the yeast enzyme from efficiently catalysing the oxidation of long-chain primary alcohols with branching at the 4 position (e.g. 4-methyl-1-pentanol, cinnamyl alcohol). This residue has been altered to Leu by site-directed mutagenesis. The alteration yields an enzyme that serves as an effective catalyst for both longer straight-chain primary alcohols and branched chain alcohols.

SYNTHESIS OF FRAGMENTS OF TRANSFORMING GROWTH-FACTOR-ALPHA INCORPORATING EXO-2-AZABICYCLO[2,2,1]HEPTANE-3-CARBOXYLIC ACIDS AS PROLINE SUBSTITUTES
Mellor, JM; Richards, NGJ; Sargood, KJ; Anderson, DW; Chamberlin, SG; Davies, DE
Tet. Lett.36(37) 6765-6768 (1995)
<Abstract>

Two novel amino acids, the enantiomers of exo 2-azabicyclo[2,2,1]heptane-3-carboxylic acid have ben independently synthesized by the aza Diels Alder reaction using a chiral auxiliary. The two acids have been advanced via solid phase synthesis to afford linear sequences, which have been cyclized to afford cyclic disulfide peptides, analogues of fragments of TGF alpha. The structures of these and other fragments have been firmly established.

Predicting the conformation of proteins from sequences - progress and future progress
Benner, SA; Jenny, TF; Cohen, MA; Gonnet, GH
Adv. Enz. Reg.34(5179) 269-353 (1994)

Analysis of amino-acid substitution during divergent evolution - the 400 by 400 dipeptide substitution matrix
Gonnet, GH; Cohen, MA; Benner, SA
Biochem. Biophys. Res. Comm.199(2) 489-496 (1994)
<Abstract>

Most formal methods for analyzing the divergent evolution of protein sequences assume a Markov model where position i in a polypeptide chain undergoes amino acid substitution independently from position i+1. The large number of aligned homologous sequence pairs available from the exhaustive matching of the protein sequence database makes it possible to examine this assumption empirically. We have constructed a 400 by 400 matrix that reports empirical probabilities for the interconversion of all pairs of dipeptides in proteins undergoing divergent evolution. Comparison of these probabilities with those expected if substitution at adjacent positions in a protein sequence were independent reveals interesting patterns that arise through the breakdown of this assumption. Several of these are useful in extracting conformational information from patterns of conservation and variation in homologous protein sequences. (C) 1994 Academic Press, Inc.

Evaluating predictions of secondary structure in proteins
Jenny, TF; Benner, SA
Biochem. Biophys. Res. Comm.200(1) 149-155 (1994)
<Abstract>

To learn how secondary structure assignments diverge during divergent evolution, pairs of proteins with solved crystal structures were aligned and their assignments compared as a function of evolutionary distance. Residues assigned in one structure to a helix or a strand are frequently paired with residues assigned in the other to a coil. However, residues assigned to a helix in one structure are almost never paired with residues assigned to a strand in the other. This suggests additional limitations to the ''three state residue-by-residue'' score commonly used to evaluate secondary structure predictions and suggests recommendations for how secondary structure predictions should be scored to assess accurately their value as starting points for modelling tertiary structure. (C) 1994 Academic Press, Inc.

Nonstandard hydrogen-bonding in duplex oligonucleotides - the base-pair between an acceptor-donor-donor pyrimidine analog and a donor-acceptor-acceptor purine analog
Voegel, JJ; Benner, SA
J. Am. Chem. Soc.116(15) 6929-6930 (1994)

Reverse transphosphorylation by ribonuclease A needs an intact p(2)-binding site - point mutations at lys-7 and arg-10 alter the catalytic properties of the enzyme
Boix, E; Nogues, MV; Schein, CH; Benner, SA; Cuchillo, CM
J. Biol. Chem.269(4), ASBMB 2529-2534 (1994)
<Abstract>

Bovine pancreatic ribonuclease A interacts with RNA along multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. This work gives additional strong support to the existence of the postulated phosphate-binding subsite p2 (Pares, X., Llorens, R., Arus, C., and Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579) and confirms the central role of Lys-7 and Arg-10 in establishing an electrostatic interaction with a phosphate group of the substrate. The effects of charge elimination by Lys-7 --> Gln (K7Q) and/or Arg-10 --> Gln (R10Q) substitutions in catalytic and ligand-binding properties of ribonuclease A have been studied. The values of K(m) for cytidine 2',3'-cyclic phosphate and cytidylyl-3',5'-adenosine are not altered but are significantly increased for poly(C). In all cases, k(cat) values are lower. Synthetic activity, i.e. the reversion of the transphosphorylation reaction, is reduced for K7Q and R10Q mutants and is practically abolished in the double mutant. Finally, the extent of the reaction of the mutants with 6-chloropurine-9-beta-D-ribofuranosyl 5'-monophosphate indicates that the phosphate ionic interaction in P2 is weakened. Thus, p2 modification alters both the catalytic efficiency and the extent of the processes in which an interaction of the phosphate group of the substrate or ligand with the p2-binding subsite is involved.

Bona fide prediction of aspects of protein conformation - Assigning interior and surface residues from patterns of variation and conservation in homologous protein sequences
Benner, SA; Badcoe, I; Cohen, MA; Gerloff, DL
J. Mol. Biol.235(3) 926-958 (1994)

Orthocyclophanes .3. Ketonands, Novel ketonic crowns of polyoxo[1(N)]orthocyclophane constitution
Lee, WY; Park, CH; Kim, HJ; Kim, SS
J. Org. Chem.59(4) 878-884 (1994)
<Abstract>

Synthetic studies of a new family of novel ketonic macrocycles are reported. Exhaustive oxidation of all of the methylenes in odd-numbered [1(n)]orthocyclophanes ([1(n)]OCPs) resulted in the polyoxo derivatives of a cyclopolyorthobenzoyl or polyoxo[1(n)]orthocyclophane constitution. This new class of ketonic crowns is referred to as [1(n)]orthocyclophanepolyones and includes [1(5)] orthocyclophane-pentaone, [1(7)]orthocyclophaneheptaone, and [1(9)]orthocyclophanenonaone. We suggest the generic name ''ketonands'' for these ketonic crowns. Structures of ketonands were confirmed by spectral and X-ray crystallographic analyses.

A prediction of the secondary structure of the pleckstrin homology domain
Jenny, TF; Benner, SA
Proteins20(1) 1-3 (1994)
<Abstract>

A consensus prediction for the secondary structure of the pleckstrin homology (PH) domain is presented. The prediction is based on an analysis of patterns of conservation and variation of homologous protein sequences. The structure is predicted to be formed largely from beta strands with a single alpha helix. (C) 1994 Wiley-Liss, Inc.

Amino acid substitution during functionally constrained divergent evolution of protein sequences
Benner, SA; Cohen, MA; Gonnet, GH
Prot. Eng.7(11) 1323-1332 (1994)
<Abstract>

In aligning homologous protein sequences, it is generally assumed that amino acid substitutions subsequent in time occur independently of amino acid substitutions previous in time, i.e. that patterns of mutation are similar at low and high sequence divergence. This assumption is examined here and shown to be incorrect in an interesting way. Separate mutation matrices were constructed for aligned protein sequence pairs at divergences ranging from 5 to 100 PAM units (point accepted mutations per 100 aligned positions). From these, the corresponding log-odds (Dayhoff) matrices, normalized to 250 PAM units, were constructed. The matrices show that the genetic code influences accepted point mutations strongly at early stages of divergence, while the chemical properties of the side chains dominate at more advanced stages.

Predicting protein crystal-structures
Benner, SA; Gerloff, DL; Jenny, TF
Science265(5179) 1642-1644 (1994)

Guanosine derivatives bearing an N2-3-imidazolepropionic acid
Heeb, NV; Benner, SA
Tet. Lett.35(19) 3045-3048 (1994)
<Abstract>

Synthesis of a T-deoxyguanosine analog tethered through the exocyclic nitrogen via a 3 carbon chain to the 4-position of an imidazole is described. The imidazole forms a hydrogen bond with the 2'-hydroxyl group of a complementary cytosine bound as a Watson-Crick base pair.

Expanding the genetic lexicon: incorporating non-standard amino acids into proteins by ribosome-based synthesis
Benner, SA
Trends Biotech.12(5) 158-163 (1994)
<Abstract>

Only 20 amino acids are normally incorporated into proteins synthesized in living cells, and this has limited the structural range of proteins that can be prepared. New methods that allow the incorporation of amino acids that are not normally encoded by natural genes are being developed: these include reassigning functions within the existing genetic code, and expanding the genetic code by constructing additional, non-natural codons. Used in conjunction with recent major advances in understanding protein structure-function relationships, these approaches should extend the range of de novo protein designs that are possible.

A secondary structure prediction of the hemorrhagic metalloprotease family
Gerloff, DL; Jenny, TF; Knecht, LJ; Benner, SA
Biochem. Biophys. Res. Comm.194(1) 560-565 (1993)

Enzymatic recognition of the base pair between isocytidine and isoguanosine
Switzer, CY; Moroney, SE; Benner, SA
Biochemistry32(39), ACS 10489-10496 (1993)
<Abstract>

The ability of various polymerases to catalyze the template-directed formation of a base pair between isoguanine (iso-G) and isocytosine (iso-C) in duplex oligonucleotides has been investigated. A new procedure was developed for preparing derivatives of deoxyisoguanosine suitable for incorporation into DNA using an automated DNA synthesizer. T7 RNA polymerase, AMV reverse transcriptase, and the Klenow fragment of DNA polymerase all incorporated iso-G opposite iso-C in a template. T4 DNA polymerase did not. Several polymerases also incorporated iso-G opposite T, presumably through pairing with a minor tautomeric form of iso-G complementary to T. In a template, iso-G directs the incorporation of both iso-C and T when Klenow fragment is the catalyst and only U when T7 RNA polymerase is the catalyst. Further, derivatives of iso-C were found to undergo significant amounts of deamination under alkaline conditions used for base deprotection after automated oligonucleotide synthesis. Both the deamination reaction of iso-C and the ambivalent tautomeric forms of iso-G make it unlikely that the (iso-C).(iso-G) base pair was a part of information storage molecules also containing the A.T and G.C base pairs found in primitive forms of life that emerged on planet earth several billion years ago. Nevertheless, the extra letters in the genetic alphabet can serve useful roles in a contemporary laboratory setting.

A word in your protein (reprinted from nature, vol 361, pg 12, 1993)
Gonnet, GH; Benner, SA
Chem. Tech.23(3) 66-66 (1993)

The nitrogenase MoFe protein - a secondary structure prediction
Gerloff, DL; Jenny, TF; Knecht, LJ; Gonnet, GH; Benner, SA
FEBS Lett.318(2) 118-124 (1993)
<Abstract>

Surface residues, interior residues, and parsing residues, together with a secondary structure derived from these, are predicted for the MoFe nitrogenase protein in advance of a crystal structure of the protein, scheduled shortly to appear in Nature. By publishing this prediction, we test our method for predicting the conformation of proteins from patterns in the divergent evolution of homologous protein sequences in a way that places the method 'at risk'.

Predicting the conformation of proteins - man versus machine
Benner, SA; Gerloff, DL
FEBS Lett.325 29-33 (1993)
<Abstract>

Two types of approaches for predicting the conformation of proteins from sequence data have lately received attention: 'black box' tools that generate fully automated predictions of secondary structure from a set of homologous protein sequences, and methods involving the expertise of a human biochemist who is assisted, but not replaced, by computer tools. A friendly controversy has emerged as to which approach offers a brighter future. In fact, both are necessary. Nevertheless, a snapshot of the controversy at this instant offers much insight into the structure prediction problem itself.

The donor-acceptor-acceptor purine analog: Transformation of 5-aza-7-deaza-1H-isoguanine (= 4-aminoimidazo-[1,2-a]-1,3,5-triazin-2(1H)-one) to 2'-deoxy-5-aza-7-deaza-isoguanosine using purine nucleoside phosphorylase
Voegel, JJ; Altorfer, MM; Benner, SA
Helv. Chim. Acta76(5) 2061-2069 (1993)
<Abstract>

A new synthesis is reported for 4-aminoimidazo[1,2-a]-1,3,5-triazin-2(1H)-one (= 5-aza-7-deaza-iso-guanosine; 8), a purine analog that, when incorporated into an oligonucleotide chain, presents a H-bond donor-acceptor-acceptor pattern to a complementary pyrimidine analog. A protected ribose derivative was coupled to 8 to yield 4-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazin-2(8H)-one (= 5-aza-7-deaza-isoguanosine; 11) after deprotection, Alternatively, direct synthesis of both the ribo derivative 11 and the corresponding deoxyribo derivative 17 as the beta-D-anomers was achieved using the enzyme purine nucleoside phosphorylase in a one-pot reaction. This adapts a known synthetic approach to yield a new strategy for obtaining diastereoisomerically pure deoxyribonucleoside analogs on 1-gram scales.

Determination of the absolute-configuration of dimethyl (2S,3S)-2-allyl-3-hydroxyglutarate - a chiral building-block for preparing branched-chain nucleoside analogs
Arslan, T; Herradon, B; Schweizer, BW; Benner, SA
Helv. Chim. Acta76(8) 2969-2975 (1993)
<Abstract>

A yeast-catalyzed reduction of dimethyl (2S,3S)-2-allyl-3-hydroxyglutarate is the key step in the preparation of bis-homo, branched-chain nucleoside analogues. To establish unambiguously the stereochemical course of the microbial reaction, the product has been converted to a derivative esterified with camphanoyl chloride, and a crystal structure of the derivative solved.

Carbocyclic analogs of nucleosides .4. preparation of enantiomerically pure analogs of purine nucleosides for the synthesis of sulfone-linked oligonucleotide analogs
Jenny, TF; Benner, SA
Helv. Chim. Acta76(2) 826-841 (1993)
<Abstract>

Cyclopentane derivatives bearing a 3-(hydroxymethyl) group, a 4-(2-hydroxyethyl) functionality, and a nucleoside base are carbocyclic variants of nucleoside analogs previously described as building blocks for the preparation of oligonucleotide analogs having dimethylene sulfone (= methanosulfonylmethano) linking groups replacing the phosphodiester linking units found in natural oligonucleotides. These carbocyclic nucleoside analogs (e.g. 17 and 20) are stable to both acid-catalyzed depurination and base-catalyzed hydrolysis, in contrast with most non-ionic analogs of oligonucleotides. Furthermore, they can be prepared with complete control over the stereochemistry at the 'anomeric' center. A procedure is given for preparing these purine-nucleoside analogs via the construction of an enantiomerically pure carbocyclic skeleton (Schemes 1-3), followed by a Mitsunobu-type reaction to introduce the purine-base derivatives (Scheme 4). Furthermore, preliminary results for the coupling of these analogs to yield nucleoside dimers (e.g. 26) are also reported (Scheme 5).

Biscyclophanes .2. Regioselectivity in the acid-catalyzed cycloalkylation of benzylbenzylic alcohol (BBA)
Lee, WY; Sim, WB; Kim, HJ; Yoon, SH
J. Chem. Soc., Perkin Trans. 1(6) 719-729 (1993)
<Abstract>

o-Benzylbenzylic alcohols (o-BBAs), in which the terminal benzyl alcohol is substituted by repeating benzyl chains all in the ortho sense, have been found to have conspicuous regioselectivity in acid-catalysed cycloalkylation, giving rise to various cyclophanes as intramolecular Friedel-Crafts alkylation products. The structure of the cyclisation products was largely dependent upon the size of the benzylic alcohols. Acidic treatment of 2-nuclear o-BBA 6 gave a [1.1]orthocyclophane 7 with a 6-membered ring, whereas 3-nuclear o-BBA 1 afforded [1.1.1]orthocyclophane 2 with a 9-membered ring in preference to a 6-membered-ring product. Higher homologues, such as 4- and 5-nuclear o-BBAs, gave rise to [1.(4)](1,2)(1,2)(1,2)(1,3)cyclophanes 14 and 25 with a 13-membered ring unit, respectively. Cyclophanes with a larger-than-1 3-membered ring have never been isolated as cycloalkylation products of o- BBA. Generalisations have been made about the priority of formation of cycles in the cycloalkylation of o-BBA in acid, to give a cycloalkylation rule, which involves the priority order of 13-membered ring > 9-membered ring > 6-membered ring. The regioselectivity was consistent with the acid-catalysed cycloalkylation of alpha,omega-benzylbenzylic diols, which yielded common-nuclear biscyclophanes. The sizes and structures of the biscyclophane products are also dependent upon the sizes and structures of the terminal benzylic diols.

Empirical and structural models for insertions and deletions in the divergent evolution of proteins
Benner, SA; Cohen, MA; Gonnet, GH
J. Mol. Biol.229(4) 1065-1082 (1993)

Predicted secondary structure for the src homology 3 domain
Benner, SA; Cohen, MA; Gerloff, D
J. Mol. Biol.229(2) 295-305 (1993)

Reduction of 2-substituted 3-oxoglutarates mediated by baker's yeast. Variation in enantioselectivity without corresponding variation in diastereoselectivity
Arslan, T; Benner, SA
J. Org. Chem.58(8) 2260-2264 (1993)
<Abstract>

The reduction of 2-substituted 3-oxoglutarates by yeast yields a new class of chiral building blocks, 2-allyl- and 2-propargyl-3-hydroxyglutarates. These are useful as starting points for the synthesis of, inter alia, branched chain analogs of sugars and nucleosides. When allyl is the side chain, the principal product has the absolute configuration (2S,3S), proven by correlation with a compound whose absolute configuration was established by crystallography. Several features of this yeast-mediated reduction are noteworthy. First, its diastereoselectivity is higher than its enantioselectivity, especially with the propargyl side chain. Further, with all substrates, variation in enantioselectivity is not manifested by a variation in diastereoselectivity. This example therefore serves as a warning for those using yeast-mediated reactions that diastereoselectivity cannot be accepted as a substitute for direct measurements of enantioselectivity, even with analogous substrates and similar reaction conditions. Finally, an unexpected metabolism of impurities in the starting material by the yeast made the overall transformation preparatively useful.

Synthesis and tautomeric equilibrium of 6-amino-5-benzyl-3-methylpyrazin-2-one - an acceptor-donor-donor nucleoside base analog
Voegel, JJ; Vonkrosigk, U; Benner, SA
J. Org. Chem.58(26) 7542-7547 (1993)
<Abstract>

6-Aminopyrazin-2-one, when incorporated as pyrimidine base analog into an oligonucleotide, might participate in a nonstandard base pair that retains a Watson-Crick geometry but is joined by a nonstandard hydrogen bonding pattern. Such base pairs can, at least in principle, be recognized independently in duplex nucleic acids. To explore the tautomeric properties that govern hydrogen bonding of this heterocycle, 6-amino-5-benzyl-3-methylpyrazin-2-one was synthesized. The equilibrium constant for the interconversion of the keto and hydroxyl tautomeric forms was estimated by comparing its ultraviolet spectrum with those of N- and 0-methyl derivatives in water, methanol, ethanol, dioxane, and water-dioxane mixtures. A plot of the logarithm of the tautomeric equilibrium constant versus Dimroth's microscopic dielectric constant (ET(30)) was linear. On the basis of an extrapolation of this relationship to the microscopic dielectric of water, 6-amino-5-benzyl-3-methylpyrazin-2-one is expected to favor at equilibrium the keto form over the hydroxyl form by a factor of ca. 2000 under conditions where DNA and RNA polymerases operate. This is substantially better than the tautomeric ratio observed with isoguanosine, where the minor form has been observed to create tautomeric ambiguity with some polymerase systems.

A word in your protein
Gonnet, GH; Benner, SA
Nature361(6408) 121-121 (1993)

Synthesis, structure and activity of artificial, rationally designed catalytic polypeptides
Johnsson, K; Allemann, RK; Widmer, H; Benner, SA
Nature365(6446) 530-532 (1993)
<Abstract>

BIOLOGICAL macromolecules with catalytic activity can be created artificially using two approaches. The first exploits a system that selects a few catalytically active biomolecules from a large pool of randomly generated (and largely inactive) molecules. Catalytic antibodies1 and many catalytic RNA molecules2 are obtained in this way. The second involves rational design of a biomolecule that folds in solution to present to the substrate an array of catalytic functional groups3-8. Here we report the synthesis of rationally designed polypeptides that catalyse the decarboxylation of oxaloacetate via an imine intermediate. We determine the secondary structures of the polypeptides by two-dimensional NMR spectroscopy. We are able to trap and identify intermediates in the catalytic cycle, and to explore the kinetics in detail. The formation of the imine by our artificial oxaloacetate decarboxylases is three to four orders of magnitude faster than can be achieved with simple amine catalysts: this performance rivals that of typical catalytic antibodies.

Reading the palimpsest: Contemporary biochemical data and the RNA world
Benner, SA; Cohen, MA; Gonnet, GH; Berkowitz, DB; Johnsson, KP
The RNA World, ed. Raymond F. Gesteland, Thomas R. Cech, and John F. Atkins, Cold Spring Harbor Laboratory Press 27-70 (1993)

Catalysis: design versus selection
Benner, SA
Science261(5127) 1402-1403 (1993)

Selective protection and deprotection procedures for thiol and hydroxyl groups
Huang, Z; Benner, SA
SynLett(1) 83-84 (1993)
<Abstract>

A protecting group strategy has been developed that permits the convergent synthesis of oligonucleotide analogs containing dimethylene sulfone groups replacing of phosphate diester groups. The strategy is based on experimental conditions that allow selective removal of dimethoxytrityl groups from oxygen and sulfur.

Evolutionary guidance in biological chemistry
Benner, SA
Biochemistry31(7), ACS 2188-2188 (1992)

MULTIDOMAIN BINDING OF TRANSFORMING GROWTH FACTOR-ALPHA TO THE EPIDERMAL GROWTH-FACTOR RECEPTOR
Richter, A; Conlan, JW; Ward, ME; Chamberlin, SG; Alexander, P; Richards, NGJ; Davies, DE
Biochemistry31(40), ACS 9546-9554 (1992)
<Abstract>

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGFalpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGFalpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGFalpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGFalpha, derived from H-1 NMR measurements [Kline, T.-P., Brown, F. K., Brown, S. C., Jeffs, P. W., Kopple, K. D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGFalpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGFalpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand. Such a model is consistent with recent investigations of the interaction of EGF-R and its ligands using physical methods, which have indicated changes in the solution conformation of the receptor upon ligand binding [Greenfield, C., Hiles, I., Waterfield, M. D., Federwisch, M., Wollmer, A., Blundell, T. L., & McDonald, N. (1989) EMBO J. 8, 4115-41231. We anticipate that the receptor-binding assay reported in this study might also be more generally applicable in probing the interaction of other biologically important peptides, and proteins, with cellular receptors of similar structure to that of EGF-R.

Carbocyclic analogs of nucleosides .2. synthesis of 2',3'-dideoxy-5'-homonucleoside analogs
Jenny, TF; Horlacher, J; Previsani, N; Benner, SA
Helv. Chim. Acta75(6) 1944-1954 (1992)
<Abstract>

A set of derivatives of cyclopentaneacetic acid cis-substituted at position 3 by nucleoside bases (both purines and pyrimidines) were prepared and characterized (see 11, 14, and 23a, b; Schemes 2-4). These molecules are carbocyclic analogs of 2',3'-dideoxy-5'-homonucleosides. In this synthesis, the skeleton was constructed from norbornanone and a novel method based on Mitsunobu chemistry used for the introduction of nucleoside-base substituents. The scope of this method was further explored via the preparation of a cyclobutyl analog of dideoxyguanosine (see 17, Scheme 3).

OXYGENATION OF COBALT(II)-SUBSTITUTED LIMULUS-POLYPHEMUS HEMOCYANIN - KINETICS, CD, AND MCD STUDIES
Larrabee, JA; Baumann, TF; Chisdes, SJ; Lyons, TJ
Inorg. Chem.31(17) 3630-3635 (1992)
<Abstract>

Cobalt(II)-substituted Limulus polyphemus (CoHcy) is characterized by circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. At neutral pH, the active site Co(II)'s are mostly aquoCoHcy, which gives rise to weak CD but intense low-temperature (4.2 K) MCD spectral features at 571, 552, and 526 nm. At higher pH's CoHcy is mostly hydroxoCoHcy and still has a weak visible CD spectrum, but three near-UV CD peaks at 372, 340, and 316 nm appear which are proposed to arise from ligand to metal charge transfer (LMCT). The 4.2 K MCD spectrum of hydroxoCoHcy is very rich with peaks at 304, 324, 355, 526, 556, 571, 616, and 642 nm. These spectra can be interpreted in terms of approximate C3, ligand symmetry about each active-site cobalt(II) with a (HiS)30 ligand set. In the case of aquoCoHcy, the 0 ligand comes from a coordinated water, whereas, in hydroxoCoHcy, the 0 ligand comes from hydroxide. The hydroxoCoHcy spectrum in the d-d transition region has too many peaks to be accounted for solely from spin-allowed transitions; therefore, it is proposed that the 616- and 642-nm bands arise from spin-forbidden 4A2 --> 2A2(G),2E(G) transitions which gain intensity from the nearby spin-allowed 4A2 --> 4E(P) and 4A2(P) transitions. The aquoCoHcy and hydroxoCoHcy MCD spectral features are strikingly similar to those of the low-pH and high-pH forms of cobalt-substituted carbonic anhydrases, respectively. HydroxyCoHcy rapidly oxygenates (k = 500 M-1 s-1) to form oxyCoHcy, which has strong CD bands at 332, 413, 518, and 618 nm. These bands are supportive of an oxyCoHcy active site, which contains mu-1,2-peroxo, mu-hydroxo dibridged Co(III) dimers. The 332- and 413-nm bands are due to pi(a)* --> d(sigma)* and pi(b)* --> d(sigma)* O2(2-) --> Co(III) LMCT, while the 518- and 618-nm CD bands are d-d transitions arising from six-coordinate Co(III).

A biomimetic biotechnological process for converting starch to fructose - thermodynamic and evolutionary considerations in applied enzymology
Moradian, A; Benner, SA
J. Am. Chem. Soc.114(18) 6980-6987 (1992)
<Abstract>

A process for preparing fructose from starch has been designed to have a thermodynamic profile similar to those found in natural metabolic pathways and implemented in a reactor containing five enzymes acting together. The process runs at equilibrium, with a final exergonic step pulling intermediates to fructose, the desired product. Therefore, the yields of fructose are high and not dominated by the glucose-fructose equilibrium constant that constrains the commercial process, which uses xylose isomerase to catalyze its final step. Three different strategies were used to find enzymes suitable for catalyzing the final irreversible step, the hydrolysis of fructose-6-phosphate: (a) recruiting an enzyme to operate backwards with respect to its physiological function; (b) recruiting an enzyme to accept a non-natural substrate through the use of a cosubstrate; and (c) developing an indirect route for converting fructose-6-phosphate to fructose. As presently implemented, the process converts starch and inorganic phosphate to glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and then fructose and inorganic phosphate; the last is recycled. The net hydrolysis of fructose-6-phosphate to yield fructose is obtained via a transaldolase-catalyzed reaction between fructose-6-phosphate and glyceraldehyde to yield fructose and glyceraldehyde-3-phosphate, which is then hydrolyzed to regenerate glyceraldehyde and inorganic phosphate using a 3-phosphoglycerate phosphatase recruited to act on an unnatural substrate. This work illustrates general ideas that may prove useful in designing other multistep biocatalytic transformations, in particular, the focus on the energetics of the pathway and on the evolution of enzymes as a guide to selecting enzymes useful in biocatalytic processes.

Ribosome-mediated incorporation of a nonstandard amino-acid into a peptide through expansion of the genetic-code
Bain, JD; Switzer, C; Chamberlin, AR; Benner, SA
Nature356(6369) 537-539 (1992)
<Abstract>

ONE serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns 1,2. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role 3-9.

Correct structure prediction
Benner, SA; Cohen, MA; Gerloff, D
Nature359(6398) 781-781 (1992)

N2-isobutyrl-O6-[2-(para-nitrophenyl)ethyl]guanine: A new building block for the efficient synthesis of carbocyclic guanosine analogs
Jenny, TF; Schneider, KC; Benner, SA
Nucl. Nucl.11(6) 1257-1261 (1992)
<Abstract>

N2-Isobutyryl-O6-[2-(p-nitrophenyl)ethyl]guanine (4) allows the synthesis of different types of carbocyclic analogs of guanosine in high yield under Mitsunobu conditions. Only the desired N-9-substituted derivatives of guanine are formed.

Exhaustive matching of the entire protein sequence database
Gonnet, GH; Cohen, MA; Benner, SA
Science256(5062) 1443-1445 (1992)
<Abstract>

The entire protein sequence database has been exhaustively matched. Definitive mutation matrices and models for scoring gaps were obtained from the matching and used to organize the sequence database as sets of evolutionarily connected components. The methods developed are general and can be used to manage sequence data generated by major genome sequencing projects. The alignments made possible by the exhaustive matching are the starting point for successful de novo prediction of the folded structures of proteins, for reconstructing sequences of ancient proteins and metabolisms in ancient organisms, and for obtaining new perspectives in structural biochemistry.

Computer-speed and sequence comparison - response
Benner, SA; Cohen, MA; Gonnet, GH
Science257(5077) 1609-1610 (1992)

Efficient regioselective synthesis of guanosine analogs
Jenny, TF; Benner, SA
Tet. Lett.33(44) 6619-6620 (1992)
<Abstract>

Reaction conditions are presented that allow regioselective introduction (N-9 versus N7) of guanine into sugar analogs under Vorbruggen conditions. Using these conditions, a set of N2-protected guanosine analogs has been prepared with N2-isobutyryl-O6-[2-(p-nitrophenyl)ethyl]guanine (1) as nucleophile. This approach helps solve an important synthetic problem in the preparation of guanosine analogs.

Patterns of divergence in homologous proteins as indicators of secondary and tertiary structure - a prediction of the structure of the catalytic domain of protein-kinases
Benner, SA; Gerloff, D
Adv. Enz. Reg.31(5010) 121-181 (1991)

A C-nucleotide base pair - methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA-polymerase
Piccirilli, JA; Moroney, SE; Benner, SA
Biochemistry30(42), ACS 10350-10356 (1991)
<Abstract>

With templates containing 2'-deoxy-1-methylpseudouridine (d(m)PSI), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

Site-directed mutagenesis of bovine pancreatic ribonuclease: lysine-41 and aspartate-121
Trautwein, K; Holliger, P; Stackhouse, J; Benner, SA
FEBS Lett.281 275-277 (1991)
<Abstract>

Chemical modification studies suggest that two residues of bovine pancreatic ribonuclease A (RNase A), Lys-41 and Asp-121, are important for catalysis. Three mutants of RNase A have been prepared, two point mutants with Lys-41 altered to Arg-41 and Asp-121 altered to Glu-121, and a double mutant where both residues are altered. The Lys-41 Arg mutant has ca. 2% the catalytic activity (k(cat)/K(m)) of the native protein, while the Asp-121 Glu mutant has ca. 17% the catalytic activity of the native protein. The double mutant has catalytic activity comparable to the Lys-41 Arg mutant.

A direct route to 3-(D-ribofuranosyl)pyridine nucleosides
Piccirilli, JA; Krauch, T; Macpherson, LJ; Benner, SA
Helv. Chim. Acta74(2) 397-406 (1991)
<Abstract>

A route for synthesizing C-nucleosides with 2,6-substituted pyridines as heterocyclic aglycones is described. Condensation of appropriately substituted lithiated pyridines with ribono-1,4-lactone derivatives yields hemiacetal 4a-g (Table 1), which can be reduced by Et3SiH and BF3.Et2O to the corresponding C-nucleoside (see Scheme 1 for 4d --> beta-D-5). Conditions are presented that optimize the amount of the 2,6-dichloropyridine-derived beta-D-anomer beta-D-5 formed (Table 3). Aminolysis of beta-D-5 yeilds the diaminonucleoside 14 (Scheme 3).

Building blocks for oligonucleotide analogs with dimethylene-sulfide, -sulfoxide, and -sulfone groups replacing phosphodiester linkages
Huang, Z; Schneider, KC; Benner, SA
J. Org. Chem.56(12) 3869-3882 (1991)
<Abstract>

Two routes are presented for the synthesis of 3',5'-bishomodeoxyribonucleosides, building blocks needed to synthesize oligodeoxynucleotide analogues where the OPO2O groups are replaced by CH2SCH2, CH2SOCH2, and CH2SO2CH2 units. Two of these have been coupled to create an uncharged analogue of a dinucleotide. As isosteric, achiral, and nonionic analogues of natural oligonucleotides stable to both enzymatic and chemical hydrolysis, such molecules have potential application as probes in the laboratory, in studies of the role of individual genes in biological function, and as ''antisense'' oligonucleotide analogues for the treatment of diseases.

Synthesis of RNA containing inosine - analysis of the sequence requirements for the 5' splice site of the tetrahymena group-I intron
Green, R; Szostak, JW; Benner, SA; Rich, A; Usman, N
Nucl. Acids Res.19(15) 4161-4166 (1991)
<Abstract>

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.

Structural determinants of stereospecificity in yeast alcohol dehydrogenase
Weinhold, EG; Glasfeld, A; Ellington, AD; Benner, SA
Proc. Natl. Acad. Sci. USA88(19) 8420-8424 (1991)
<Abstract>

Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its k(cat) value and makes one stereochemical "mistake" every 850,000 turnovers (instead of almost-equal-to 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a co-factor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochemical "mistake" every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochemical fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis.

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate-specificity?
Allemann, RK; Presnell, SR; Benner, SA
Prot. Eng.4(7) 831-835 (1991)
<Abstract>

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3' --> 5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.

RNA world
Benner, SA; Ellington, AD
Science252(5010) 1232-1232 (1991)

Carbocyclic analogs of nucleosides via modified Mitsunobu reactions
Jenny, TF; Previsani, N; Benner, SA
Tet. Lett.32(48) 7029-7032 (1991)
<Abstract>

A set of carbocyclic nucleoside analogs have been prepared using a novel modification of the Mitsunobu reaction. This approach helps solve an important synthetic problem in the preparation of carbocyclic analogs of nucleosides.

The ribonuclease from an extinct bovid ruminant
Stackhouse, J; Presnell, SR; Mcgeehan, GM; Nambiar, KP; Benner, SA
FEBS Lett.262(1) 104-106 (1990)

Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease
Schein, CH; Haugg, M; Benner, SA
FEBS Lett.270 229-232 (1990)

Oligonucleotides containing flexible nucleoside analogs
Schneider, KC; Benner, SA
J. Am. Chem. Soc.112(1) 453-455 (1990)

The stereospecificities of seven dehydrogenases from Acholeplasma laidlawii - the simplest historical model that explains dehydrogenase stereospecificity
Glasfeld, A; Leanz, GF; Benner, SA
J. Biol. Chem.265(20), ASBMB 11692-11699 (1990)

Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet
Piccirilli, JA; Krauch, T; Moroney, SE; Benner, SA
Nature343(6253) 33-37 (1990)

Progenote or protogenote
Benner, SA; Ellington, AD
Science248(4958) 943-944 (1990)

Building-blocks for oligonucleotide analogs with dimethylene-sulfide, dimethylene-sulfoxide, and dimethylene-sulfone groups replacing phosphodiester linkages
Schneider, KC; Benner, SA
Tet. Lett.31(3) 335-338 (1990)

Patterns of divergence in homologous proteins as indicators of tertiary and quaternary structure
Benner, SA
Adv. Enz. Reg.28 219-236 (1989)

Enzyme kinetics and molecular evolution
Benner, SA
Chem. Rev.89(4) 789-806 (1989)

The stereospecificity of the ferrous-ion-dependent alcohol-dehydrogenase from zymomonas-mobilis
Glasfeld, A; Benner, SA
Euro. J. Biochem.180(2) 373-375 (1989)

An improved system for expressing pancreatic ribonuclease in Escherichia coli
Mcgeehan, GM; Benner, SA
FEBS Lett.247(1) 55-56 (1989)

Enzymatic incorporation of a new base pair into DNA and RNA
Switzer, C; Moroney, SE; Benner, SA
J. Am. Chem. Soc.111(21) 8322-8323 (1989)

Modern metabolism as a palimpsest of the RNA world
Benner, SA; Ellington, AD; Tauer, A
Proc. Natl. Acad. Sci. USA86(18) 7054-7058 (1989)

The return of pancreatic ribonucleases
Benner, SA; Allemann, RK
Trends Biochem. Sci.14(10) 396-397 (1989)

Stereospecificity in enzymology - its place in evolution
Benner, SA; Glasfeld, A; Piccirilli, JA
Topics Stereochem.19(3) 127-207 (1989)

Extracellular 'communicator RNA'
Benner, SA
FEBS Lett.233(2) 225-228 (1988)

Crystal-structures of 2 simple n-substituted dihydronicotinamides - possible implications for stereoelectronic arguments in enzymology
Glasfeld, A; Zbinden, P; Dobler, M; Benner, SA; Dunitz, JD
J. Am. Chem. Soc.110(15) 5152-5157 (1988)

Stereochemical profile of the dehydrogenases of Drosophila melanogaster
Allemann, RK; Hung, R; Benner, SA
J. Am. Chem. Soc.110(16) 5555-5560 (1988)

The design of synthetic genes
Presnell, SR; Benner, SA
Nucl. Acids Res.16(5) 1693-1702 (1988)

Return of the 'last ribo-organism'
Benner, SA; Ellington, AD
Nature332(6166) 688-689 (1988)

Interpreting the behavior of enzymes. Purpose or pedigree?
Benner, S. A., Ellington, A. D
Crit. Rev. Biochem. Mol. Biol. (1988) 23, 369-426

Anomeric specificity of L-fucose dehydrogenase - a stereochemical imperative in aldopyranose dehydrogenases
Berkowitz, DB; Benner, SA
Biochemistry26(9), ACS 2606-2611 (1987)

Redesigning life - organic-chemistry and the evolving protein
Benner, SA
Chimia41(5) 142-148 (1987)

Natural selection, protein engineering, and the last riboorganism: Rational model building in biochemistry
Benner, SA; Allemann, RK; Ellington, AD; Ge, L; Glasfeld, A; Leanz, GF; Krauch, T; Macpherson, LJ; Moroney, S; Piccirilli, JA; Weinhold, E
Cold Spring Harb. Symp. Quant. Biol.52(2) 53-63 (1987)

Expression of bovine pancreatic ribonuclease A in Escherichia-coli
Nambiar, KP; Stackhouse, J; Presnell, SR; Benner, SA
Euro. J. Biochem.163(1) 67-71 (1987)

The stereospecificity of oxaloacetate decarboxylase - a stereochemical imperative
Piccirilli, JA; Rozzell, JD; Benner, SA
J. Am. Chem. Soc.109(26) 8084-8085 (1987)

Free energy differences between enzyme bound states
Ellington, AD; Benner, SA
J. Theoret. Biol.127(4) 491-506 (1987)

The last ribo-organism
Benner, SA; Ellington, AD
Nature329(6137) 295-296 (1987)

Evolution guidance - engineering alcohol-dehydrogenase and ribonuclease
Weinhold, EG; Ellington, A; Presnell, SR; Mcgeehan, GM; Benner, SA
Prot. Eng.1(3) 236-237 (1987)

Benner's model - a successful prediction
Benner, SA
Chem. Eng. News64(4) 3-3 (1986)

Body-temperature and the specific-heat of water
Dunitz, JD; Benner, SA
Nature324(6096) 418-418 (1986)

Dynamic transduction of energy and internal equilibria in enzymes: A reexamination of pyruvate kinase
Stackhouse, J; Nambiar, KP; Burbaum, JJ; Stauffer, DM; Benner, SA
J. Am. Chem. Soc.107(9) 2757-2763 (1985)

A stereochemical imperative in dehydrogenases: New data and criteria for evaluating function-based theories in bioorganic chemistry
Benner, SA; Nambiar, KP; Chambers, GK
J. Am. Chem. Soc.107(19) 5513-5517 (1985)

Stereochemical imperative in enzymic decarboxylations - stereochemical course of the decarboxylation catalyzed by acetoacetate decarboxylase
Rozzell, JD; Benner, SA
J. Am. Chem. Soc.106(17) 4937-4941 (1984)

Total synthesis and cloning of a gene coding for the ribonuclease-s protein
Nambiar, KP; Stackhouse, J; Stauffer, DM; Kennedy, WP; Eldredge, JK; Benner, SA
Science223(4642) 1299-1301 (1984)

A mechanistic basis for the stereoselectivity of enzymatic transfer of hydrogen from nicotinamide cofactors
Nambiar, KP; Stauffer, DM; Kolodziej, PA; Benner, SA
J. Am. Chem. Soc.105(18) 5886-5890 (1983)

Combining enzymatic and chemical steps in the synthesis of biochemically valuable compounds: Isotopically chiral methyl acetate
Rozzell, JD; Benner, SA
J. Org. Chem.48(8) 1190-1193 (1983)

The stereoselectivity of alcohol dehydrogenases - a stereochemical imperative
Benner, SA
Experientia38(5) 633-637 (1982)

Rearrangement of a geometrically restricted triepoxide to the first topologically nonplanar molecule: A reaction-path elucidated by using oxygen isotope effects on carbon-13 chemical shifts
Benner, SA; Maggio, JE; Simmons, HE
J. Am. Chem. Soc.103(6) 1581-1582 (1981)

Optical activity due to isotopic substitution. Vibrational circular dichroism and the absolute configurations of α-deuterated cyclohexanones
Polavarapu, PL; Nafie, LA; Benner, SA; Morton, TH
J. Am. Chem. Soc.103(18) 5349-5354 (1981)

Analogs for acetoacetate as an enzyme substrate - stereochemical preferences
Benner, SA; Morton, TH
J. Am. Chem. Soc.103(4) 991-993 (1981)

Stereospecificity and stereochemical infidelity of acetoacetate decarboxylase (AAD)
Benner, SA; Rozzell, JD; Morton, TH
J. Am. Chem. Soc.103(4) 993-994 (1981)

Diphenylphosphinodithioic acid - a reagent for the conversion of nitriles to thioamides
Benner, SA
Tet. Lett.22(20) 1851-1854 (1981)

Mechanism of the reaction of diphenylphosphinodithioic acid with nitriles
Benner, SA
Tet. Lett.22(20) 1855-1858 (1981)

Preparation of an ecdysone immunogen for radioimmunoassay work
Hung, DT; Benner, SA; Williams, CM
J. Biol. Chem.255(13), ASBMB 6047-6048 (1980)

The Tricarboxylic Acid Cycle
Steven Benner
Yale Scientific (1976) 50 (7), 4-10, 34-36

Asymmetry in organic chemistry. A left-handed molecule looks at the world.
Steven Benner
Yale Scientific (1976) 50 (February), 16-20

Sailing from Genoa.
Steven Benner
Yale Scientific (1976) 50 (7), 2-3

Enzyme mechanism
Steven Benner
Yale Scientific (1975) 49 (October), 4-8

The Sahel famine. A crisis of technology misuse.
Steven Benner
Yale Scientific (1975) 49, 2-20

Energy crisis
Steven Benner
Yale Scientific (1974) 48 (4), 10-15