FFAME.org :: Steven Benner's Publications

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. USA (2010) [Epub ahead of print] PMID: 20080675

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. acids 28 (4) 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.

Signatures of a Shadow Biosphere
Davies, PCW Benner, SA Cleland, CE Lineweaver, CH McKay, CP Wolfe-Simon, F
Astrobiology 9 (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.

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.

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. acids 27 (3) 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. acids 27 43-56 (2008)

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)

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)

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

Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins
Bradley, ME Benner, SA
BMC Bioinformatics 7 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 Genomics 7 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.

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.

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. acids 25 (12) 1309-1333 (2006)
<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.

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.

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.

Planetary systems biology
Benner, SA Ricardo, A
Mol. Cell 17 (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.

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.

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.

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.

Quantitative analysis of a RNA-cleaving DNA catalyst obtained via in vitro selection
Carrigan, MA Ricardo, A Ang, DN Benner, SA
Biochemistry 43 (36) 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.

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 Biology 2 (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.

Expanding the genetic alphabet: Pyrazine nucleosides that support a donor-donor-acceptor hydrogen-bonding pattern
von Krosigk, U Benner, SA
Helv. Chim. Acta 87 (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.

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).

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.

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.

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.

Borate minerals stabilize ribose
Ricardo, A Carrigan, MA Olcott, AN Benner, SA
Science 303 (5655) 196-196 (2004)

Redesigning genetics
Benner, SA
Science 306 (5296) 625-626 (2004)

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

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
Astrobiology 3 (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
Biochemistry 42 (28) 8598-8598 (2003)

Evolutionary, structural and biochemical evidence for a new interaction site of the leptin obesity protein
Gaucher, EA Miyamoto, MM Benner, SA
Genetics 163 (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.

A direct synthesis of nucleoside analogs homologated at the 3 '- and 5 '-positions
Schmidt, J Eschgfaller, B Benner, SA
Helv. Chim. Acta 86 (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. Acta 86 (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.

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
Nature 421 (6919) 118-118 (2003)

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins
Gaucher, EA Thomson, JM Burgan, MF Benner, SA
Nature 425 (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.

C-5 modified nucleosides: Direct insertion of alkynyl-thio functionality in pyrimidines
Held, HA Roychowdhury, A Benner, SA
Nuc. Nuc. Nuc. acids 22 (4) 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.

Nucleobase pairing in Watson-Crick-like genetic expanded information systems
Geyer, CR Battersby, TR Benner, SA
Structure 11 (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.

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).

From phosphate to bis(methylene) sulfone: Non-ionic backbone linkers in DNA
Hutter, D Blaettler, MO Benner, SA
Helv. Chim. Acta 85 (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.

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)

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.

The past as the key to the present: Resurrection of ancient proteins from eosinophils
Benner, SA
Proc. Natl. Acad. Sci. USA 99 (8) 4760-4761 (2002)

Evolution - Planetary biology - Paleontological, geological, and molecular histories of life
Benner, SA Caraco, MD Thomson, JM Gaucher, EA
Science 296 (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
Science 297 (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.

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
Nature 409 (6819) 459-459 (2001)

Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors
Gaucher, EA Miyamoto, MM Benner, SA
Proc. Natl. Acad. Sci. USA 98 (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 Genetics 17 (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)

Synthesis and characterization of oligonucleotides containing 2 '-deoxyxanthosine using phosphoramidite chemistry
Jurczyk, SC Horlacher, J Devined, KG Benner, SA Battersby, TR
Helv. Chim. Acta 83 (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.

Evaluation measures of multiple sequence alignments
Gonnet, GH Korostensky, C Benner, S
J. Comp. Bio. 7 (1-2) 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.

The missing organic molecules on Mars
Benner, SA Devine, KG Matveeva, LN Powell, DH
Proc. Natl. Acad. Sci. USA 97 (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.

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
Science 290 (5496) 1506-1506 (2000)

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 (1-2) A24-A24 (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. C 123 (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.

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) A1415-A1415 (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. Acta 82 (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.

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.

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.

The molecular origins of life - Assembling pieces of the puzzle
Benner, SA
Science 283 (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)

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
Biochemistry 37 (12) 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
Biochemistry 37 (12) 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.

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.

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. Acta 81 (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.

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)

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. acids 17 (5) 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.

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 Moroney, SE Muller, E Nambiar, KP Piccirilli, JA Switzer, CY Vogel, JJ Richert, C Roughton, AL Schmidt, J Schneider, KC Stackhouse, J
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
Syn. Lett. (6) 688-U264 (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.

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) A1295-A1295 (1997)

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)

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. USA 94 (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
Proteins 27 (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
Proteins 27 (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
Syn. Lett. (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.

Catalysis: The omnipresent phenomenon.
Benner, SA
FASEB J. 10 (6) C2-C2 (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.

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. Acta 79 (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. Acta 79 (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.

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.

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
Science 274 (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.

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. B 112 (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)

Reconstructing ancient forms of life
Benner, SA
JOURNAL OF CELLULAR BIOCHEMISTRY (19) 200-200 (1995)

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
Nature 374 (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
Nature 377 (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. USA 92 (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
Proteins 21 (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
Proteins 21 (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
Proteins 22 (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
Proteins 23 (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.

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) 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)

A prediction of the secondary structure of the pleckstrin homology domain
Jenny, TF Benner, SA
Proteins 20 (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
Science 265 (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
Biochemistry 32 (39) 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 (1-2) 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. Acta 76 (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. Acta 76 (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. Acta 76 (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).

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
Nature 361 (6408) 121-121 (1993)

Synthesis, structure and activity of artificial, rationally designed catalytic polypeptides
Johnsson, K Allemann, RK Widmer, H Benner, SA
Nature 365 (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
Science 261 (5127) 1402-1403 (1993)

Selective protection and deprotection procedures for thiol and hydroxyl groups
Huang, Z Benner, SA
Syn. Lett. (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
Biochemistry 31 (7) 2188-2188 (1992)

Carbocyclic analogs of nucleosides .2. synthesis of 2',3'-dideoxy-5'-homonucleoside analogs
Jenny, TF Horlacher, J Previsani, N Benner, SA
Helv. Chim. Acta 75 (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).

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
Nature 356 (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
Nature 359 (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
Science 256 (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
Science 257 (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
Biochemistry 30 (42) 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 (1-2) 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. Acta 74 (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. USA 88 (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
Science 252 (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 (1-2) 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) 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
Nature 343 (6253) 33-37 (1990)

Progenote or protogenote
Benner, SA Ellington, AD
Science 248 (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 (1-2) 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. USA 86 (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
Nature 332 (6166) 688-689 (1988)

Anomeric specificity of L-fucose dehydrogenase - a stereochemical imperative in aldopyranose dehydrogenases
Berkowitz, DB Benner, SA
Biochemistry 26 (9) 2606-2611 (1987)

Redesigning life - organic-chemistry and the evolving protein
Benner, SA
Chimia 41 (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
Nature 329 (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. News 64 (4) 3-3 (1986)

Body-temperature and the specific-heat of water
Dunitz, JD Benner, SA
Nature 324 (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
Science 223 (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
Experientia 38 (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) 6047-6048 (1980)