Steven A. Benner - US grants

Hypotheses are proposed as solutions to one of the most prominent stereochemical problems in enzymology: why some NAD(P)+-dependent dehydrogenases transfer exclusively the pro-R (A) hydrogen of NAD(P)H while others transfer exclusively the pro-S (B) hydrogen of NAD(P)H. The explanation for the stereoselectivities observed is based on a combination of proposed structural and thermodynamic properties of nicotinamide cofactors and the enzymes that use them, and suggests new ideas for understanding stereoselectivity, catalysis, and evolution in enzymes in general. This proposal seeks funding to test these hypotheses, and will use the hypotheses as a guide to the study of the stereochemical and thermodynamic properties of nicotinamide cofactors and dehydrogenases. Physical techniques (nmr, thermodynamic measurements), enzymological methods (kinetics, stereochemical studies), and combinations of the two will be used. The understanding of enzymes and their catalytic abilities that this work should produce is fundamental for understanding biological systems and disease processes.

We have shown that a consensus secondary structure for a family of proteins can be accurately predicted from a set of aligned homologous protein sequences using heuristics that extract conformational information from patterns of conservation and variation from the alignment, provided that these heuristics are based on an accurate understanding of how protein sequences divergently evolve under functional constraints. We will to continue to develop these heuristics for predicting the secondary, supersecondary, and tertiary structure of proteins starting from sequence alignments. This work will test heuristics already successfully used to make bona fide predictions of secondary structure by systematic and fully automated application against proteins with known structure. These heuristics will be made available to the public via a server accessible through -electronic mail. Efforts will then be directed towards creating "perfect" secondary structure prediction that are compelling starting points for modelling tertiary structure. A detailed analysis will be made of the remaining misassignments made using our heuristics, which fall into five classes: (a) errors where conformation within a family of proteins has diverged, (b) errors where the multiple alignment is bad, (c) misassignments of internal helices, (d) misassignment of secondary structure near the active site, and (e) misassignments of surface beta strands. Modified heuristics will be developed to eliminate each class of misassignment, or to ensure that misassignments do not disrupt efforts-to model tertiary structure starting from a predicted secondary structure. "Core" and "non-core" secondary structural elements will be defined and prediction heuristics scored by their relative ability to detect core elements. A systematic study of internal helices will be undertaken to refine techniques that have successfully identified an internal helix in the hemorrhagic metalloproteinases. A systematic study of secondary structure near the active site will be undertaken, to learn more about how peptide conformation in these regions might be modelled. Tools for identifying joints between domains, long distant compensatory covariation, and "parsing" strings will be developed. A database of surface beta strands will be constructed and analyzed to learn more about how these might be distinguished from surface coils. Advanced statistical methods (GOR, neural networks) as well as our heuristics will then be comparatively evaluated, with the goal of obtaining a composite prediction tool that combines the best elements of each method. This grant will also permit us to continue make bona fide predictions. In a second set of studies, we will continue our work analyzing patterns of amino acid substitution during divergent evolution under functional constraints, implementing our most advanced substitution matrices to improve pairwise alignments. We will then develop tools for detecting distant homologies, both through the reconstruction of probabilistic ancestral sequences in a protein family, and through the comparison of predicted secondary structures of separate protein families.

Heuristics have been developed that allow the prediction of the secondary structure of proteins starting from a set of aligned homologous protein sequences. These heuristics extract conformational information from patterns of conservation and variation within the family of proteins. The tools as presently implemented involve both automated and manually applied tools. They have been tested by making bona fide prediction of protein secondary structure, those announced before an experimental structure becomes available, for approximately a dozen proteins. These predictions have proven to be remarkably accurate. Further, they have defined a few secondary structural element types that are difficult to predict, thus focusing future work. Thus, they are a significant step towards meeting one goal of the National Library of Medicine to develop "algorithms capable of predicting structure [of proteins] based on primary sequences of amino acids" (Item 103.D). The work to be funded under this proposal will transfer from Switzerland to the United States these prediction technologies when the Principal Investigator moves to the University of Florida in 1995. The proposed work will test the feasibility of preparing computer software that can be used on workstations to automate those aspects of the structure prediction tools that are presently applied by hand. PROPOSED COMMERCIAL APPLICATION: Tools that enable the prediction of the folded structure of proteins from sequence data are commercially marketable as computer software, as well as core units in drug discovery and drug development programs.

DESCRIPTION (verbatim from the applicant's abstract): The goal of this research, chosen by the BNP Panel from a palette of science presented in the previous submission, is "to contribute to a fine-grained understanding of interactions" between polymerases (pols) and their substrates. We will: (1) synthesize nucleotide analogs that form Watson-Crick pairs with different hydrogen bonding patterns, C-glycosides, without an unshared electron pair (UEP) in the minor groove, with functionality in the major groove, with substantial contributions from minor tautomeric equilibria, and able to form purine-purine and pyrimidine-pyrimidine mismatches; (2) Mutate DNA pols and reverse transcriptases to develop enzymes better able handle unnatural nucleotides; and (3) Measure the ability of pols to incorporate these analogs to test hypotheses concerning their interaction between pols and their substrates. The technological goals are stated as a proposition: Should a biological chemist wish to put an unnatural nucleotide into a DNA molecule via template-directed polymerization, this research will identify the natural pol to do this best, suggest ways to mutate the pols to do it better, and suggest alternative forms of the nucleotide that are easier to incorporate. Three hypotheses will be tested: Hypothesis 1: Can answers to these questions predict how an unnatural nucleotide will interact with a pol? (a) Does the nucleotide have an unshared pair of electrons in the minor groove? (b) Is it a C-glycoside or an N-glycoside? (c) Does it present a large substituent to the major groove (d) What is the ratio of its tautomeric forms? (e) Can it form purine-purine and pyrimidine-pyrimidine mismatches joined by three hydrogen bonds? Hypothesis 2: Is the evolutionary history of a pol the best predictor of how it interacts with unnatural nucleotides? Hypothesis 3: To modulate the interaction between a pol and a particular nucleotide, are amino acids in the second and third "Shell," 10-18 A away from the active site Mg, the relevant ones to change? This project fits our long term goal in organic chemistry: to develop an artificial genetic system based on an expanded genetic information systems (AEGIS), and our overall vision for the future of molecular science, where evolutionary theory from biology will be joined to structure theory from chemistry, joining the two principal traditions in science, natural history and the physical sciences, in a way that permits the power of each to contribute to the nation's biomedical research needs.

DESCRIPTION: (Applicant's abstract) The long-term aim of this work is to develop evolution-based technologies for the study of biological function in open reading frames (ORFs) identified by genome projects. The specific aims of this proposal are to: 1. Implement structure prediction tools to identify long distance homologs in genomic databases. This is the first step towards assigning function to an open reading frame (ORF) with unknown function. Prediction of secondary structure is a powerful tool for identifying long distance homologies that cannot be detected by simple comparison of sequence data. Because structure prediction requires sequence data as the only input, it is a low cost way of expanding the value of existing genome sequence databases. 2. Implement tools for reconstructing the evolutionary history of nuclear families of proteins. The tools will identify in the evolutionary history of nuclear families of proteins episodes of divergent evolution of a type that indicates conserved function, and other episodes of divergent evolution of a type that characterizes divergence without function. These tools will permit the user to assess the likelihood that the function of a homologous protein is the same as that of an ORF obtained from the genome database. This tool also requires only sequence data as input, making it another low cost way of expanding the value of genome sequence databases. 3. Develop tools for correlating episodes of functional evolution in protein families with geological periods where new physiology emerged in metazoan animals. These tools will allow a user to generate hypotheses relating protein evolution to possible functions of ORFs. This tool draws on existing and developing paleontological databases, and couples sequence data directly to biological function. These tools will be implemented within Darwin, a comprehensive platform for performing theoretical and practical genomic analysis. Further, they will directly interface with the new field of experimental paleobiochemistry, which permits the experimental testing of hypotheses relating function and behavior derived from the tools described above.

DNA and RNA are virtually unique among polymers in organic chemistry to display role-base self-assembly over a broad range of molecular structure. This self-assembly is the basis of biotechnology and genetics, and is the focus of this proposal. Work in the Benner laboratories has shown that the Watson-Crick base pair can support 12 nucleobases joined in six distinct base pairs, each joined by different patterns of hydrogen bonding, to give An Expanded Genetic Information System (AEGIS) following an expanded set of Watson- Crick pairing rules. AEGIS can be copied via template-directed polymerization catalyzed by DNA and RNA polymerases. Work in the Tan laboratories has developed and exploited near-field scanning optical microscopy (NSOM) tools to image single molecules with 10- 20 nm resolution on two-dimensional surfaces, in particular, nanostructure functional probes having the dimensions of single molecules. Using these technologies, Tan has imaged a variety of molecules and nanostructures (buckminsterfulleranes, DNA chains, and cell membranes), photonanofabricated a variety of biochemically functionalized nanostructures, analyzed the kinetics of single enzyme molecules, and probed the release of metabolites inside single cells. The two research groups, housed in adjacent buildings, therefore have technologies ideally matched to undertake a collaborative project focusing on the practical aspects of synthesis/fabrication through bio-self assembly of nanostructures. Procedures for synthesizing the components of the nanostructures using biocatalysts (in particular, thermostable DNA polymerases) will be explored, with an eye towards in situ generation of the components of nanostructures. The cells of the largest nano-nets will be loaded with photoreactive groups that can be written/read by NSOM. We hope that this will create a small readable/writable memory element with obvious relevance to practical applications.

DESCRIPTION: The goal of this grant is to develop a new strategy for joining physiological function to biochemical behavior in proteins. This approach combines protein structure, mechanism, and engineering with evolutionary analyses. Central to this analysis is the reconstruction of evolutionary history of the protein superfamily from sequence data, and preparing ancient proteins from extinct organisms in the laboratory where they can be studied. The evolution of molecular and biological behavior is set in a historical context by correlating it with the evolution of organisms (deduced from paleontology) and the surrounding ecology, allowing construction of testable hypotheses concerning the structure-behavior relationships in this family, mechanisms by which these compounds exert their biological activities, and possible physiological function(s) of the proteins, as well as allowing to engineer new proteins with desired behaviors. The work here will use ribonuclease as a system to develop this strategy. Bovine pancreatic RNase A belongs to a superfamily of proteins that has, in various members, evolved to an enormous range of interesting biological behaviors. RNase homologs are known that are immunosuppressive, block the growth of tumor cells (But not normal cells), kill Schistosoma and Trichinella, cause neurological damage, cause lung damage in asthmatic patients, display lectin-like behavior, inhibit infection of mammalian cells by HIV-1, or do none of these. These behaviors have medical relevance; several RNase variants are now in clinical and preclinical stages of testing for their useful biomedical activities. In the next phase of this ongoing research program, the physiological significance of biological behaviors of seminal RNase will be assessed, a crystal structure solved to determine the mechanism by which seminal RNase binds and melts duplex DNA, the impact of the introduction of cysteines on the kinetics of folding and the thermodynamics of the folded structure will be examined and new sequence data will be collected to complete the model of the evolutionary history of this important class of molecules. This study will show how new biomolecular function is created in higher organisms, by mutation, insertion, deletion gene duplication and recruitment of duplicate genes. This evolutionary approach differs from that pursued in other laboratories, and this work will continue to develop a new paradigm where evolutionary information is integrated with chemical and biological information to solve biomedical research problems in a "post-genome" environment.

DESCRIPTION (provided by applicant): The DNA alphabet is not limited to the four standard nucleotides. Rather, twelve nucleobases forming six base pairs joined by mutually exclusive hydrogen bonding patterns are possible within the geometry of the Watson-Crick base pair. These form An Expanded Genetic Information System (Aegis), a new, "rule based" molecular recognition system that carries protein functionality, can be prepared in combinatorial form, and can be copied much like nucleic acids. Given the rich understanding of this system after a decade of research in the Benner laboratory, Aegis is a technology transfer opportunity. In the Phase I study, a comprehensive set of experiments validated the ability of Aegis to provide a set of orthogonal, non-cross reacting tags useful to support highly multiplexed (100 or more analytes) DNA diagnostics systems. These included studies in both solution and solid phase, with the Luminex instruments that support multiplexing, and with polymerases. In Phase II, we will prepare 1000 independently binding DNA-like tags, further develop tools to support genotyping of individual patients, and select for polymerases that incorporate both functionalized nucleotides and Aegis components into oligonucleotides. PROPOSED COMMERCIAL APPLICATION: Chemical systems that support "rule based molecular recognition" have wide commercial applicability, including in come "blue skies" (nanotechnology and molecular computing, for example). More immediately, the Aegis system here is supporting a commercial diagnostics system, and has led in the Phase I period to an assay that detects single nucleotide polymorphisms (SNPs). Such assays promise a revolution in predictive medicine and personalized care, with potential market value.

DESCRIPTION (Applicant's abstract): A substantial commercial potential exists for software tools that allow a biomedical research scientist to use genomic data to form experimentally testable hypotheses. These will be used to exploit genomic sequence data to understand the aetiology of disease, to improve diagnostic tools, and to develop more effective therapies. The Master Catalog, a commercial product developed jointly by EraGen Biosciences and the Benner laboratory at the University of Florida, provides a convenient framework for implementing heuristics that do this. The Master Catalog is a naturally organized database that contains evolutionary trees, multiple sequence alignments, and reconstructed evolutionary intermediates for all of the proteins in the GenBank database. The Benner laboratory has developed and anecdotally tested heuristics that date events in the molecular history, provide evidence for and against functional recruitment within a protein family, detect distant homologs, associate individual residues important for functional changes with a crystal structure, find metabolic and regulatory pathways, and correlate events in the molecular record with the history of life on Earth. This Phase I proposal seeks to validate a set of these heuristics more broadly to determine their suitability for database-wide application. In Phase II, we will implement these within the Master Catalog, and launch a commercial bioinformatics product to support functional analysis of genomic databases. PROPOSED COMMERCIAL APPLICATION: In its present version, the Master Catalog is a successful commercial product within a niche: "best in class" of bioinformatics databases. Adding a validated set of heuristics for extracting functional information from genome databases will make it the software of choice for most functional genomics work, and be a central tool in the pharmaceutical/ biotechnology industries. Academic versions and student versions will find markets in most universities.

Professor Benner will lead a project that combines organic synthesis, biological chemistry, in vitro evolution (lVE) and univariate statistical analysis that will develop the physical organic chemical tools needed to quantitate the impact of adding chemical functionality (amino, thiol, imidazole, peptides, and metal chelating groups) via covalent attachment to catalytic DNA molecules. This work will first address the paradox that arises from the observation that endowing DNA and RNA libraries with additional functionality evidently improves the catalytic potential of nucleic acids by only a factor of two to ten, not by the orders of magnitude expected from standard Structure Theory in Organic Chemistry. A set of five hypotheses that account for the orders-of-magnitude discrepancy between expectation and observation will be tested, including models that hold (a) that our view of the role of functionality in catalysis is naive, (b) that the functional endowment of natural DNA (phosphates and hydrogen bond donating and accepting groups) are sufficient for catalysis in general, (c) that the functionality recruited non-covalently by nucleic acids from solution (in particular, divalent cations such as Mg++) overwhelms the contribution of covalently linked functionality, (d) that lVE experiments lose the best catalysts, and (e) that more than one type of functional group is needed before the expected large benefit from functionality is seen. This will require careful assessment of the kinetic order of the reaction being effected by the selected DNA molecules (first order, unimolecular, with the catalytic step rate determining?).

With this Award, the Organic and Macromolecular Chemistry Program (OMC) and the Molecular Biochemistry Program in the Division of Molecular and Cellular Biosciences (MCB) will support the research of Professor Steven A. Benner of the University of Florida. Professor Benner's work is expected to have broad impact. From a practical perspective, we may learn how to truly get "catalysis on demand" from IVE experiments, useful for everything from biomedicine to environmental remediation. From a scientific perspective, we will understand in greater depth the possibilities of single biopolymer systems playing a role in the origin of life. From a methodological perspective, he will develop tools that permit IVE to explore the distribution of chemical properties in "structure space" defined by a DNA sequence. And, from a theoretical perspective, the work may end up altering, perhaps dramatically, our global view of the relationship between functionality and reactivity in nucleic acids.

This Nanoscale Interdisciplinary Research Team (NIRT) award supports a group of five faculty, including four at the University of Florida and one at Cornell University, to develop tools capable of measuring the distribution and concentration of specific messenger RNA molecules (mRNAs) in defined subcellular regions of single nerve cells. Initial effort will use neurons from the model organism, Aplysia. Use of the tools will then be extended to neurons from higher organisms with the goal of understanding how neurons establish new connections or synapses. Using electron beam technology, the team will fabricate one dimensional (1-D) DNA nanoarrays for the capture and direct assay of the mRNAs. Detection will employ molecular beacons to generate a fluorescent signal in the presence of specific target mRNAs; the beacons are fluorescent nanoparticles consisting of self-assembling branched DNA nanostructures designed using an artificially expanded genetic alphabet (AEGIS). Nanofluidics and dip-pen nanolithography will be tasked with delivery of the nanoparticles to specific sites in the DNA array. Fluorescence will be detected by optical imaging. Software specialized for analyzing biological molecules will archive and interpret recovered mRNA sequences using interpretive proteomics tools developed from evolutionary models. The project will benefit from a collaborative setting where students at all levels engage multiple disciplines. If successful, this will provide an educational paradigm for the training of the scientists of the future, as well as demonstrating the utility of nanoscience and engineering in the study of classical problems in biology.

DESCRIPTION (provided by applicant): Target Assisted Combinatorial Synthesis (TACS) is a new method to identify pharmacophores, collections of functional groups that confer binding ability to a specific target, and from there to develop lead compounds for drug development.. In a TACS experiment, a therapeutic target is presented with a library of n fragments prepared by convergent chemical synthesis. Composites of these fragments are then formed by reaction between the reactive groups of two or more fragments in the library under conditions of dynamic equilibrium. The target then selects the tightest binding composite (the preferred composite) from a pool of n2 composite compounds prepared in situ. With 104 to 105 (n) ligand fragments in a typical library, the effective library sizes explored in a TACS experiment are 108-1010 compounds (n2) in a "two-fold compositing" TACS experiment, and still more when three or more fragments are assembled in a composite. The TACS method thereby allows exploration of vast regions of structure space for pharmacophores and their combinations, yet prepares analyzable quantities of fragments, and does not require significantly more target than a classical screening or combinatorial experiment directed against a library supported on beads. Supporting the analysis of TACS experiments is Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS), stored waveform inverse Fourier transform (SWIFT), and infrared multiphoton laser disassociation (IRMPD). Together, these generate information about the structure of a target-bound composite in a single experiment. The goal of this proposal is to develop the TACS strategy to the point where it is selected by medicinal chemists for targets for which it is appropriate, and use TACS to address several issues in Diversity Science. We target two families of biological molecules of biomedical interest: (a) the Src homology 2 (SH2) domain family, whose members are important in cell regulation, cancer, and a variety of other diseases; (b) the eosinophil cationic proteirdeosinophil-derived neurotoxin (ECP/EDN) family, which plays key roles in allergy, anaphylaxis, and inflammation. Fragment libraries include trialkoxyphosphines, diols, and diamines, which will be prepared using phosphite ester chemistry, metathesis, thiamin condensation and peptide chemistry. Compositing chemistry will involve the coordination of trialkoxyphosphines and diamines with metals, and diols with borates. These activities will generate non-peptidic lead compounds that bind to SH2 domains, non-nucleic acid lead compounds that bind to human EDN/ECP, a general, empirically based view of the scope and limitations of the TACS strategy, and a deeper understanding of structure and behavior of large diverse collections of small organic molecules.

DESCRIPTION (provided by applicant): Target Assisted Combinatorial Synthesis (TACS) is a new method to identify pharmacophores, collections of functional groups that confer binding ability to a specific target, and from there to develop lead compounds for drug development.. In a TACS experiment, a therapeutic target is presented with a library of n fragments prepared by convergent chemical synthesis. Composites of these fragments are then formed by reaction between the reactive groups of two or more fragments in the library under conditions of dynamic equilibrium. The target then selects the tightest binding composite (the preferred composite) from a pool of n2 composite compounds prepared in situ. With 104 to 105 (n) ligand fragments in a typical library, the effective library sizes explored in a TACS experiment are 108-1010 compounds (n2) in a "two-fold compositing" TACS experiment, and still more when three or more fragments are assembled in a composite. The TACS method thereby allows exploration of vast regions of structure space for pharmacophores and their combinations, yet prepares analyzable quantities of fragments, and does not require significantly more target than a classical screening or combinatorial experiment directed against a library supported on beads. Supporting the analysis of TACS experiments is Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS), stored waveform inverse Fourier transform (SWIFT), and infrared multiphoton laser disassociation (IRMPD). Together, these generate information about the structure of a target-bound composite in a single experiment. The goal of this proposal is to develop the TACS strategy to the point where it is selected by medicinal chemists for targets for which it is appropriate, and use TACS to address several issues in Diversity Science. We target two families of biological molecules of biomedical interest: (a) the Src homology 2 (SH2) domain family, whose members are important in cell regulation, cancer, and a variety of other diseases; (b) the eosinophil cationic proteirdeosinophil-derived neurotoxin (ECP/EDN) family, which plays key roles in allergy, anaphylaxis, and inflammation. Fragment libraries include trialkoxyphosphines, diols, and diamines, which will be prepared using phosphite ester chemistry, metathesis, thiamin condensation and peptide chemistry. Compositing chemistry will involve the coordination of trialkoxyphosphines and diamines with metals, and diols with borates. These activities will generate non-peptidic lead compounds that bind to SH2 domains, non-nucleic acid lead compounds that bind to human EDN/ECP, a general, empirically based view of the scope and limitations of the TACS strategy, and a deeper understanding of structure and behavior of large diverse collections of small organic molecules.

[unreadable] DESCRIPTION (provided by applicant): This project, as its R21 milestone, will deliver Taq DNA polymerases that catalyze the template-directed addition of nucleoside triphosphates carrying large fluorescent groups attached to their 3'-ends. The fluorescent groups therefore both terminate transiently the growth of the oligonucleotide chain, and signal the nature of the nucleotide that was last added. These polymerase variants will form the core of a "cheap reagent" approach to the Sequencing by Synthesis (SbS) strategy. Gaining control over polymerase behavior is key for this approach to generate inexpensive genome-quality sequence data. The research will exploit a decade of experience in the Benner laboratory with nucleic acid analogs, polymerases that accept them, and practical application of the combination. The tactics assume that site directed and mutagenesis is generally site-directed damage, and therefore must be followed by directed evolution to obtain polymerase substrate combinations that meet specifications. Here, directed evolution will be used to restore catalytic power and fidelity in polymerases that have been engineered to accept fluorescent tags. We shall: (a) synthesize nucleoside triphosphates that have fluorescent blocking groups; (b) use directed evolution system in water-in-oil emulsions to select polymerases that accept the triphosphates efficiently and faithfully; (c) obtain polymerases to incorporate these to within 10%, the catalytic activity of native polymerases, and with specificity to better than one part in 10,000. Should this R21 milestones be passed, we will use the R33 year to develop a working prototype for a multiplexed sequencing-by-synthesis device using these polymerases. The Aims will be to: (d) optimize the fluorescent compound-cleavage chemistry-polymerase combination, (e) use an artificially expanded genetic information system (AEGIS), the artificial alphabet invented in the Benner group, to bin primer-template combinations for parallel sequencing; and (f) exploit 2D gels to develop an architecture for a prototype parallel sequencing instrument based on the technologies developed in Aims 1-3 two dimensional arrays and in gels. [unreadable] [unreadable]

With this Chemical Bonding Center (CBC) Phase I, Step II award, the Division of Chemistry and the Office of Multidisciplinary Activities of the Mathematical and Physical Sciences Directorate jointly support the research of Jack W. Szostak, of Massachusetts General Hospital, Steven A. Benner, of the University of Florida, and Gerald F. Joyce, of the Scripps Research Institute. This CBC will pursue the long-term goal of synthesizing artificial chemical systems that will exhibit Darwinian evolution. In phase I of the project, a combination of molecular design and laboratory selection will be used to generate RNA-like structures that undergo self-reproduction with heritable mutation. The CBC will utilize the synthetic organic skills of Benner, the expertise in RNA-like systems of Joyce, and the experience of Szostak in the design of in vitro evolution experiments in this highly collaborative endeavor. The synthesis of Darwinian chemical systems will form the basis of a synthetic biology that has the potential of constructing self-replicating systems using the tools of preparative chemistry. This program at the interface of chemistry and biology will provide broad interdisciplinary training for students and is expected to capture the imagination of the public.

Chemical Bonding Centers are designed to focus innovative collaborative efforts that address a "big problem" which will lead to a major advance in chemistry or at the interface of chemistry and other sciences and will have the potential to attract broad scientific and public interest.

[unreadable] DESCRIPTION (provided by applicant): This project, as its R21 Phase I milestone, will deliver a combination of conical nanopores having read length dimensions slightly less than 1 nm, and nucleobase-modified DNA oligonucleotides, where the passage of the DNA through the nanopore proceeds with a time constant of 10-100 microseconds per nucleotide, and where the ion current through the nanopore, during the time when the DNA is in transit, varies detectably depending on the nucleotide that is in the pore at the time that the current is measured. This nanopore-modified DNA combination will form the core of an extremely inexpensive technology to generate long reads of DNA sequence at the single molecule level. The research will exploit a decade of experience in the Martin laboratory preparing nanopores and engineering their chemical context, and an equal experience in the Benner laboratory working with nucleic acid analogs, polymerases that accept them, and practical applications of the combination. As specific aims, we shall: (a) prepare the nanotubes; (b) attach chemical functionality to the nanotubes; (c) prepare nucleoside triphosphates carrying different sized polyether dendrimers attached at the 5-position (for pyrimidines) and the 7-position (for 7-deazapurines); (d) use these triphosphates to synthesize modified DNA molecules. The nanopores will then be physically characterized to determine their ion transport dynamics, and in conjunction with the modified oligonucleotides, to find a combination that meets the R21 milestone specifications. If this milestone is passed, the R33 period will be used to develop sequence specific and randomly targeted primers that incorporate DNA, PNA, and tags that exploit an artificial genetic alphabet, and to develop improved processes for generating conical nanopores in a form suitable for large scale application. These will then be targeted against specific sequences extracted from mammalian genomes. [unreadable] [unreadable]

Any system, natural or man-made, can be better understood when its structure and history are considered together. Living systems are not exceptions. Nevertheless, over the past century, the molecules and cells that form the nanoscopic and microscopic structures within organisms have generally been studied separately from the histories of the organisms themselves. This has deprived biology, both as a science and a business central to biotechnology and medicine, of certain power that might come from tools that consider together its molecules, organisms, ecosystems and histories.

This NSF OPUS project will exploit recent advances in chemistry, biology and geology, together with two decades of work in Dr. Benner's laboratory, to combine these disciplines in a vertical synthesis. The project will produce tools for the researcher to facilitate this synthesis in the laboratory, including databases that historically organize the human genome, computational tools to detect molecular adaptation, and experimental strategies to test historical hypotheses in the laboratory. The project will also deliver instructional materials for students who wish to participate in this synthesis.

Many diseases, including hypertension and diabetes, reflect imperfect adaptation of our genome to rapid changes in the environments experienced by our recent primate ancestors. Many complications of advanced cancers, including those of the evolutionarily ""modern"" prostate and breast, involve the adaptation of cells via molecular change in response to medical intervention. Much data in molecular biology, from the crystal structures of proteins to the human genome, does not convey any particular meaning in the absence of a historical context. For these and other reasons, the synthesis that will be advanced through this OPUS project should have impact throughout biology, pure and applied.

DESCRIPTION (provided by applicant): This proposal concerns the artificially expanded genetic information system (AEGIS), a DNA-like molecular system having 8 letters in addition to the 4 in natural DNA. AEGIS displays five features of a synthetic genetic system (rule-based design, orthogonality, semi-predictable affinity, higher information density, and polymerase incorporability) that permits it to solve a range of problems in medical diagnostics, systems biology, and genomics. Today, over 400,000 patients have their health care improved due to AEGIS -enabled clinical assays that support personalized medicine. This project will move beyond AEGIS as a passive binding system to allow AEGIS components to participate as part of a dynamic system that exploits DNA polymerases and reverse transcriptases. This requires that we develop polymerases that incorporate AEGIS components with very high fidelity and efficiency. To obtain these, DNA polymerase that already do this reasonably well will be placed under selective pressure in a directed evolution experiment. The polymerases that are obtained will then be used in real time PCR instruments to develop nucleotide-polymerase-recipe combinations that support 6-letter polymerase chain reactions. Supporting technology, including the determination of fidelity of incorporation of these. We will then do a demonstration project to show that AEGIS components can solve a difficult problem in modern molecular, systems, and clinical biology: the multiplexing of PCR. Multiplexed PCR is the rate limiting step in many genomics analyses. Our long term, highly ambitious goal is to develop a "synthetic biology", a chemical system capable of Darwinian evolution, based on AEGIS components.

[unreadable] DESCRIPTION (provided by applicant): The proposed research will develop dynamic combinatorial chemistry as a new and innovative strategy to allow a DNA or RNA target in a biological sample to assemble its own template under conditions of dynamic equilibrium. This chemistry incorporates element that allows the template-created primer to primes the synthesis of a strand of DNA complementary to the target. The selectivity of the process is such that a single DNA sequence in a genome as complex as the human genome can be targeted. The architecture of the process, however, allows it to discriminate against single nucleotide changes with the effectiveness of small duplexes. Consistent with the basic research/business mission/plan of the Foundation for Applied Molecular Evolution and Firebird Biomolecular ("We do not make DNA assays. We develop chemistry that makes DNA assays better"), this will create a platform technology that meets specifications that are unavailable at the present time: selectivity of a probe as if it were a long oligonucleotide, but discrimination by the probe as if it were a short oligonucleotide. This platform technology should have application in many assays, including in human diagnostics and research markets. In preliminary studies, a proof of concept showed that this combinatorial chemistry architecture met the novel specifications when the target was DNA. The Phase 1 milestone will be passed if similar success can be obtained with RNA as a target. Potential commercial applications in human medicine are substantial, starting with applications in biomedical research, where these tools will allow us to detect messenger RNA, RNAi, and other RNA molecules important in biology. Several important diagnostics and drug development tools been driven in the past decade through the creation of platform chemical technologies by scientists at the Foundation for Applied Molecular Evolution. Such technologies have commercial impact far beyond their development costs because they deliver a set of chemical performance specifications that is unavailable by existing chemistry. The proposed work will develop another platform technology, this one allowing the detection of nucleic acid targets through a novel combination of dynamic combinatorial chemistry, enzymology, and nucleic acid science. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal, targeted for the National Human Genome Research Institute, has the goal of funding an R01 program to develop near term technologies to lower the cost of DNA sequencing. As illustrated by the history of DNA synthesis, costs will most likely drop through a stream of "small step" innovations in chemistry, enzymology, and instrumentation. This proposal, from a laboratory with a track record of innovation in nucleic acid chemistry, enzymology and bioinformatics, will provide this stream, in part by combining individual innovations that have emerged from the Benner laboratories, including nucleoside analogs and polymerases that accept them. The innovations are: Artificially expanded genetic information systems (AEGIS), a DNA-like system that forms duplexes following Watson-Crick rules, but has no interaction with natural nucleic acids [Ben04]. A self-avoiding molecular recognition system (SAMRS), a DNA-like molecule whose components do bind natural DNA, but do not bind other components of the same unnatural SAMRS system [Ben07b]. DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS components. Emulsion-based directed evolution (EBDE) to optimize polymerases that incorporate unnatural nucleotides. Reversible terminators that terminate primer extension, but reversibly, and polymerases that accept them. SNAP2, which allows priming of DNA synthesis with the specificity of a 16mer, but discrimination of 8mers. Naturally organized genomic databases that support primer design and help analyze the biological meaning of sequence data collected. These technologies will be combined to develop (a) SNAP2 primers that incorporate SAMRS components, permitting highly multiplexed priming and PCR with primers having an effective length of 16mers (or longer) without creating PCR artifacts and supporting single nucleotide discrimination, (b) AEGIS tags appended to SAMRS primers that allow binning of the non-repeating elements of a patient's genome on an array without the need for single molecule chemistry, and (c) DNA polymerases that accept as substrates triphosphates that combine a fluorescent tag on their gamma phosphorus units with a 3'-ONH2 reversible terminator. We will benchmark the new combination reagents and their corresponding enzymes to optimize rates of addition of triphosphates in template-directed polymerization reactions, as well maximize discrimination and minimize mismatching. Throughout, we will use the POSaM DNA array synthesizer as a "beta test " platform to examine combination reagents in the context of small-scale "sequencing-during synthesis " tests. The deliverables will be reagents that meet the "wish lists" of NHGRI with respect to cost, helping meet is $100,000 genome sequence goal, with reduced costs likely as further incremental improvements in the chemistry follow. PROJECT HEALTH RELEVANCE The NHGRI needs to have a laboratory developing, in an R01 format, new chemical reagents and enzymes to support sequencing-during-synthesis and other parallel sequencing architectures that will help it achieve is $100,000 cost-per-genome goal. It is unreasonable to expect that a single technological advance will achieve this goal in a single stroke;this is illustrated by experience in the history of DNA synthesis, where costs dropped through innovations in chemistry, enzymology, and instrumentation that continued for decades. Indeed, much of the cost that will be borne by NHGRI centers with sequencing architectures that are existing or under development comes from their application of off-the-shelf reagents rather than innovative sample processing chemistry, much of which is current available, at least in the development stage. This proposal, from a laboratory with a track record of innovation in structures, reagents, and enzymes to manipulate nucleic acids, is focused on near term development of these to enable NHGRI cost-reduction sequencing goals.

DESCRIPTION (provided by applicant): It is axiomatic that biological systems can be better understood if we understand both their structure and their histories. This proposal, directed towards the NIAAA and NIEHS, will provide the first example where historical biology is applied to an area of interest to these institutes: the evolution of the response of primates to environmental ethanol. Using an innovative combination of molecular evolution, paleontology, organic chemistry, kinetics, molecular biology, biotechnology, and crystallography, this research will yield a model that describes, from the biomolecule to the pathway, the adaptive response of primates, including humans, as they encountered, managed, and ultimately exploited a new environmental toxin, ethanol, over the past 100 million years. Our work will focus on the evolution of the alcohol dehydrogenase-aldehyde dehydrogenase (ADH-AlDH) system in primates. These enzymes form a two-step pathway that yields acetate from ethanol. Genes for these enzymes hold genetic variation in human populations that correlates with many alcohol-related diseases. We will first collect primate sequences to enrich the evolutionary models for these two superfamilies of proteins, including trees, alignments, ancestral sequences, and computational analyses of functional change within these superfamilies. These will be followed by paleogenetic experiments, where ancestral ADHs and AlDHs from human ancestors and relatives will be resurrected for study in the laboratory. Detailed analyses of substrate specificity and kinetic power will let us determine whether our ancestors followed "avoidance", "accommodation", or "utilization" strategies to manage ethanol when it first emerged, and thereafter as ethanol increased and decreased in the ecosystems of primates, until the present. These will be supplemented by analyzing the evolution of the "systems biology" properties of the system. The results will help us better understand the meaning in human biology of data collected in model organisms (e.g. rat, fly), which are separated from humans by hundreds of millions of years. Finally, we will use reductionist science, including protein crystallography, to describe at a molecular level what Darwinian processes did to manage this environment-genomics dynamic. This research will be the first collaboration between Steven Benner, who initiated experimental paleogenetics as a field and has developed planetary and systems biology in many biomolecular systems [Ben02], and Thomas Hurley, who has comprehensively studied human ADHs and AlDHs [Hur01]. In addition to producing a combined historical and reductionist analysis of this system, this work will provide a paradigm showing how this combination can be applied throughout biomedical research, and therefore have an impact on nearly every system of interest to human biology. Although it is axiomatic that diseased and healthy biology can be better understood if we understand its natural history, historical science has had difficulty entering the mainstream of biomedical research funding. This proposal, directed towards the NIAAA and NIEHS, seeks funding to support a collaboration between two laboratories to develop a detailed historical model for the evolution of the alcohol dehydrogenase-aldehyde dehydrogenase (ADH-AlDH) system in primates and closely related mammals. By combining natural history and reductionist science, the work will show how the substrate specificities and catalytic activities of these two enzymes co-evolved in response to changing environmental conditions, as the exposure of this environmental toxin changed. This will provide the first paradigm applying evolutionary analysis to an important medical problem, thereby encouraging the application of such analyses throughout medical research, where they are expected to have significant impact wherever they are applied.

DESCRIPTION (provided by applicant): This project, targeted for the National Human Genome Research Institute, will apply nearly a dozen new technologies to improve the performance of DNA chip microarrays as they detect, quantitate, and characterize nucleic acids. The technologies include: An artificially expanded genetic information system (AEGIS), a DNA-like molecular recognition system that forms duplexes following Watson-Crick rules, but does not interact with natural nucleic acids. A self-avoiding molecular recognition system (SAMRS), whose components do bind to natural DNA but do not bind to other components of the same unnatural SAMRS system. DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS Reversible terminators, which terminates primer extension but (unlike dideoxynucleoside triphosphate terminators) can be later removed to continue primer extension, and polymerases that accept them. These support "sequencing using cyclic reversible termination" to validate array hits while they are still on the-array. SNAP2 technology that primes DNA and RNA with the specificity of a 16mer but the discriminatory power of 8mers [Lea06]. Novel technology for on-array DNA synthesis using an inkjet DNA array synthesizer. Convertible nucleotides that enable downstream cloning and sequencing of DNA and RNA containing unnatural building blocks. Dendrimeric structures that perform in sandwich assays to increase the number of fluorescent tags bound to an array by an individual target analyte. To validate these technologies for on-array use, the following specific aims will be met: 1. We will use the ink jet DNA array synthesizer to prepare target arrays incorporating these technologies, establishing a cycle of synthesis-test-evaluate-redesign-resynthesis that will allow in-house benchmarking of the innovative chemistries with respect to their ability to improve sample preparation and array performance. 2. We will synthesize a range of compounds needed to support the new technologies. 3. We will benchmark the new technologies with respect to their ability to improve: 3.1 Rates of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.2 Sensitivity of hybridization of analytes to arrays independent of the complexity of the hybridization assay mixture. 3.3 Selectivity with respect to single nucleotide mismatches. 3.4 Uniformity of response of multiple array elements targeted against different regions of a single analyte. 3.5 Sensitivity of detection of low abundance analyte targets, increased using dendrimeric structures. 4. We will then adopt innovative technologies to append AEGIS tags to natural samples in various sample preparation architectures 5. Last, we will develop a system for on-array validation of positive "hits" using sequencing using reversible terminators. PUBLIC HEALTH RELEVANCE This project, targeted for the National Human Genome Research Institute, is premised on the fact that innovative new chemistries are needed to improve the performance of array-based analysis of nucleic acids to meet the demanding specifications of biomedical researchers who do genomic science, demands that have increased as the performance of microarrays has improved. The Benner laboratory has invented approximately a dozen new chemical technologies that can be applied to each step of the genomic analysis, from sample preparation to array analysis. This proposal seeks funds to benchmark the improvements in array performance generated by these new technologies, improve them by a cycle of test and redesign, and provide them to the genomics community.

DESCRIPTION (provided by applicant): Sequencing during synthesis (SdS) is an architecture for massively parallel DNA sequencing that has the strong potential of lowering the cost of sequencing enough to allow individuals access to their own genetic heritage as a way of personalizing their medical care. Thus far, however, higher-throughput next-generation sequencing systems are relatively expensive, have relatively long run times and produce relatively short reads, thereby limiting their use for diagnostic applications. This proposal will support the transfer of reagent technology from the Foundation for Applied Molecular Evolution (FfAME) to Intelligent Bio-Systems (IBS), where it will be optimized to yield, at the end of the Phase 2 period, a high-performance prototype instrument that better meets the needs of both the research and healthcare communities. PUBLIC HEALTH RELEVANCE: Sequencing during synthesis (SdS) is an architecture for doing massively parallel DNA sequencing that has the strong potential of lowering the cost of sequencing enough to allow individuals access to their own genetic heritage as a way of personalizing their medical care. Intelligent Bio-Systems (IBS) seeks to become the cost and quality leader in this next generation of DNA sequencing technology. This proposal will support the transfer of reagent technology from the Foundation for Applied Molecular Evolution (FfAME) to IBS, where it will be optimized for the IBS instrument to yield, at the end of the Phase 2 period, a commercial instrument.

DESCRIPTION (provided by applicant): In 2010, the Benner group announced the development of four innovations relevant to tools to detect human immunodeficiency virus (HIV) in complex biological samples: (a) An artificially expanded genetic information systems (AEGIS) that supports six nucleotide PCR, allowing independent amplification of small amounts of HIV RNA without interference from other DNA in the environment. (b) A self-avoiding molecular recognition system (SAMRS) that supports essentially unlimited multiplexing in DNA probing, priming, and multiplexed PCR amplification. (c) Procedures that convert standard DNA into AEGIS-containing DNA, supporting downstream orthogonal capture that allows DNA-targeted assays to be flexible and adaptive, possibly allowing new targets to be added to a multiplexed assay kit without demanding a reworking of the parts of that kit already targeted. (d) Reversible terminators that, as triphosphates, are hypothesized to allow detection and relative quantitation of variant HIV sequences. We hypothesize that by combining these innovations, we can improve HIV diagnostics tools, expanding their power to detect fewer virions in more complex biological environments with greater dynamic range and greater subtype specificity, together greater multiplexing. Further, these technologies should deliver flexibility; it should be possible to rapidly add capabilities to detect new variants, co-incident infectious agents, or even identify previously unknown variants at specific sites in the HIV genome in the course of diagnosing HIV infections. To test this hypothesis, we will perform a staged series of assay development, adding each of these innovations in series to increasingly challenging problems in the detection of HIV target sequences, starting with singleplexed detection of single HIV targets in relatively simple environments, adding innovations as we lower the amount of target molecules, increase the level of multiplexing, and make the environment more complex. At each stage, we will drive the system to fail, and note the parameters (sensitivity, complexity, multiplexing level) at which the system fails. These define a parameter space which provides a metric for progress. This project will also make available as deliverables kits of primers, probes, and detection capture beads, to be provided HIV researchers interested in benchmarking or using them. Although technology from the Benner laboratory stands behind the branched DNA (bDNA) 3.0 tool now widely used to measure HIV viral load, this is the first time that the Benner laboratory has sought funding for AIDS research. Thus, a further goal of this work will be to allow innovations from the Benner laboratory to be more widely used to solve the many HIV-related problems at the NIAID. This will help the NIAID help meet the goal established by the National HIV/AIDS Strategy of increasing the awareness of HIV status from 79% to 90% by 2015 in the US. PUBLIC HEALTH RELEVANCE: Accurate HIV diagnostic testing continues to pose challenges, and nearly 20% of the 1.1 million individuals infected with HIV are unaware of their infection. Last year, the Benner laboratory developed four innovations in DNA/RNA chemistry that are hypothesized to be able to improve sensitivity, enhance low cost multiplexing, and detect emerging variants of HIV in complex biological mixtures. This proposal seeks funding to test those hypotheses, and to deliver laboratory-ready mixtures to support analysis of HIV and its variants in those mixtures

Overview:
The 2014 Gordon Research Conference (GRC) on the Origins of Life will bring together researchers from diverse backgrounds to discuss topics aimed at understanding the origin of life on Earth and its potential distribution throughout the universe. It brings together biologists, geologists, chemists, physicists, astronomers and practitioners of numerous other disciplines from a wide variety of geographical locations. Speakers include a wide spectrum of individuals from universities, research institutes, and government organizations. The meeting will be held January 12-17th at the Hotel Galvez in Galveston, Texas.

Intellectual Merit:
The 2014 meeting is organized by Dr. Steven A. Benner (The Foundation for Applied Molecular Evolution, Gainesville, FL) and Dr. Stephen Freeland (Institute for Astronomy, University of Hawaii, HI). Both organizers are well respected in the Origins of Life community and represent several disciplines included in the meeting's scientific agenda. Topics of discussion will include: the nature and experimental reconstitution of the earliest cells; the study of organic compounds in extraterrestrial environments and their possible contribution to the prebiotic inventory, the nature of the early Earth and other planetary environments, self-replicating chemical systems, the origin and evolution of the translation apparatus and the search for life beyond Earth. The Gordon Research Conferences have a long history of providing a venue for scientific exchange among junior investigators and senior scientists in small, intimate meetings. The format of the GRC itself will follow an established pattern that includes formal talks, posters, and ample time for informal discussion. These meetings foster an atmosphere of intense scientific interaction and many opportunities for building scientific networks. Compared to other meetings on the origin of life, this meeting fills a unique niche in two respects: it has a narrower scientific focus than many larger meetings (International Conference on the Origin of Life, Bioastronomy, Astrobiology), and the U.S. location allows facile participation by young scientists who frequently cannot afford to attend international meetings abroad.

Broader Impacts:
In addition to the scientific discourse, the 2014 GRC will also explore the role of philanthropy in Big Science and how private entrepreneurs can better interact with scientists. Improving science literacy among the public will also be discussed including methods of how to effectively transfer science into science fiction in popular culture. A large effort is being made to increase the participation of women and minority groups at the meeting, including serving as discussion leaders and speakers. A concerted effort is being made to advertise the meeting to national and international organizations, including former attendees of GRC and Gordon Research Seminars (GRS) series in this and other disciplines, and members of the major international astrobiological societies.

DESCRIPTION (provided by applicant): Scientists, clinician, and physicians alike have long wanted a technology that can routinely deliver molecules that bind to targets important to their research, to diagnostic biomarkers, and to molecules essential to the progression of patient diseases. Currently, macromolecular binding molecules on demand (BMODs) are most rapidly available by way of antibody technology. Today, antibodies are gaining niches among therapeutic agents, previously dominated by small molecules generated using the hard slog of medicinal chemistry. A quarter century ago, scientists suggested that the replicability and evolvability of DNA and RNA (xNA) might offer an alternative route to macromolecular BMODs. Here, xNA aptamers might be selected from libraries of xNA molecule to bind to a target via in vitro selection (SELEX). Aptamers might work under conditions where antibodies do not, especially in environments where proteins unfold. They might eventually displace antibodies or become therapeutic agents, as are many antibodies today. Despite the successes of SELEX, we now understand that the four-nucleotide xNA that it uses has too few functional groups, too little sequence diversity, and too much interference from natural xNA, to meet this vision in its broadest form. Therefore, we propose here to expand SELEX using an artificially expanded genetic information system (AEGIS), a kind of DNA that adds up to eight independently pairing nucleotides to the four found in standard DNA. The proposed work will immediately add two AEGIS nucleotides to SELEX (Z and P, forming a Z:P pair independent of standard C:G and T:A pairs), allowing us to immediately use AEGIS-SELEX to create GACTZP aptamers that bind to lung, breast, and liver cancer cells. Immediate progress is possible because the two collaborating applicant laboratories (Steven Benner at the FfAME and Weihong Tan at UF) have already combined breakthroughs in cell-SELEX, polymerase technology, and AEGIS sequencing, to produce the first AEGIS aptamer. Obtained in just weeks from only 12 rounds of SELEX, this 30 namomolar aptamer offers up the central hypothesis for this work: Because AEGIS xNA libraries have richer diversity, they are richer reservoirs of high affinity aptamers than standard xNA libraries. By allowing xNA aptamers to gain up to 60% of the sequence diversity of antibodies, AEGIS-SELEX further offers the opportunity to finally meet the technological goals of SELEX. AEGIS-SELEX should also help expand the science of protein-nucleic acid interactions and molecular recognition in new directions. To achieve this vision, three things must be done, all shown to be feasible by preliminary work: (1) We must improve the fidelity of polymerases that copy AEGIS DNA. (2) We must improve sequencing technology for AEGIS DNA. (3) We must add more AEGIS nucleotides to the Z and P that have already been proven; and (4) we must compare AEGIS-SELEX to standard SELEX. Following a two for the price of one strategy, we will do this benchmarking by creating useful aptamers that target circulating liver, breast, and lung cancer cells.

Our view of life is very much influenced by the life on Earth around us. All of the life on Earth (that we know of) descends from a single common ancestor, and it all has fundamentally the same biochemistry. This means that our view of biology is very much biased, reflecting only what we know of our surrounding biosphere and ourselves. The scientific goal of this project is to expand this view, something important to understand the intimate connection between the phenomenon that we know as life and the molecules that underlie it. This expanded view will be important, for example, should we encounter alien life in our search of the cosmos. It is equally important if we wish to use life-like processes in commerce, manufacturing, and medicine. The "grand challenge" to be met by this project is to create a cell that is able to use an entirely synthetic form of DNA; one built from 6 nucleotide "letters", which is two more than the number of nucleotides found in natural DNA. In pursuing this challenge, more will be learned about the chemistry of metabolism, reproduction, and genetic regulation. In addition to its scientific interest as a second example of a genetic system able to evolve, the cell will also have practical value. This cell will be used to manufacture molecules for human and animal diagnostics, for homeland security, and for manufacturing as a bio safe platform. Undergraduate students will participate in the research activities, and the findings will continue to provide useful insights and perspectives on the origins of life and the nature of extraterrestrial life. This project is co-funded by the Systems and Synthetic Biology Cluster within the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes in the Chemistry Division.

This project will engineer a strain of E. coli ("Firebug") that can propagate plasmid DNA carrying nucleotides from a "second-generation" artificially expanded genetic information system (AEGIS). In its broadest form, AEGIS increases the number of independently replicable nucleotides in DNA from 4 to 12. This project will begin by adding just two AEGIS components, named (Z and P), to the four natural nucleotides (GACT), exploiting prior work that has (1) optimized second-generation AEGIS components for their chemical and enzymatic performance; (2) developed in vitro molecular biology to support AEGIS DNA, including six-letter GACTZP PCR, sequencing, and endonucleases, ligases, and kinases that use GACTZP DNA; (3) demonstrated in vitro evolution of GACTZP DNA, including generation of aptamers by evolution in the laboratory; and (4) engineered kinases that convert dZ and dP to their triphosphates inside cells. To pass the next milestone in synthetic biology that makes the expanded code functional in the cell, Firebug will be constructed. This will involve managing E. coli's error-prone polymerases and repair systems, moving enzymes encoding biosynthesis of Z and P into the chromosome, and placing the engineered cells under selection to improve their fidelity of replication of plasmid-borne Z:P pairs.

? DESCRIPTION (provided by applicant): The public health challenges from RNA viruses are at a tipping point, with dengue moving up Florida, human transmission of chickungunya now established in Florida, and, transmitted by different mechanism, Ebola. While these have long been a challenge, their incidence has been predominantly overseas. In addition, the health hazards of other RNA viruses better known in the United States remains unabated. Eastern equine encephalitis is again emerging in New England, with its 30% mortality rate. West Nile virus continues as an important health hazard. Further, given the speed of international travel and the ease with which the sequences of RNA viruses mutate, a public health official can easily have a mosquito that carries an RNA virus from one of these classes that is nevertheless not detected by PCR with standard primers. This creates a need for exactly the product that Firebird's technologies enable. These technologies are: 1. Self-avoiding molecular recognition systems (SAMRS). SAMRS primers do not interact with each other. This allows unlimited multiplexing of PCR. Further, SAMRS allows additional targets to be added to an assay without the multiplex collapsing. SAMRS also suppresses primer-dimers in isothermal amplification (e.g. RPA, HDA), making possible (in Phase 2) point of sampling kits. SAMRS therefore supports a highly adaptable molecular diagnostics able to detect dozens of RNA species for essentially the cost of detecting one. 2. Artificially expanded genetic information systems (AEGIS). AEGIS adds nucleotides to the four in standard DNA and RNA (collectively xNA). Thus, AEGIS generates primers that cannot complement any natural DNA, no matter how complex the sample, supporting assays with very low noise and very few false positives. 3. Universal base technology (Biversals). RNA viruses easily mutate; exemplifying the consequences to public health, recent surveillance of HIV-infected patients with the goal of measuring incidence found that the viruses in ~ 20% of the isolates were not PCR amplified by standard primers. Firebird's evolutionary analyses show that mutations often occur in silent sites in coding regions. Thus, Firebird scientist invented biversal nucleobases that prime on either A or G (the Y-biversal), or on either T or C (the R-biversal) Phase 1 will generate a kit to detect 22 common mosquito-borne RNA viruses, allowing public health staff to screen on a Luminex platform for all of these for less than $50, less than the cost of detecting any two RNA viruses separately. In Phase 2, these will be moved to simpler platforms. Firebird's pipeline investments allow us to prepare AEGIS and SAMRS building blocks in multi-gram amounts, and AEGIS and SAMRS oligonucleotides on demand. Because surveillance assays are not heavily regulated, and since its customers for those kits (public health staff) are well trained, this product fits well within Firebird's current product line. However, strong performance of a low cost, highly multiplexed, easily adaptable, and low false-positive surveillance product will undoubtedly help Firebird raise capital for patient-targeted applications of these technologies.

This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences and the Molecular Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Steven Benner of The Westheimer Institute for Science and Technology (TWIST) within the Foundation for Applied Molecular Evolution to expand the power of biologically created proteins. The next generation of biotechnology will use components of living systems to create unnatural biopolymers, including proteins that contain unnatural amino acids. This award will develop this biotechnology, at the same time as deepening our understanding of how proteins are made in living cells. Proteins produced by biotechnology find use in products as diverse as pharmaceuticals, cosmetics, industrial manufacture, and laundry detergents. Adding unnatural amino acids to these may open up for exploration a horizon of proteins having properties that natural proteins cannot reach. The project trains undergraduate students in methods of cutting-edge technology development.

The experiments designed for this project combine chemical synthesis with molecular biology, laboratory evolution, and bioinformatics to gain control over each step in the in vitro synthesis of proteins. This includes developing new ways to put unnatural amino acids onto unnatural transfer RNA molecules by directed evolution of charging catalysts, chemically synthesizing new nucleic acid analogs to manage infidelity in RNA biosynthesis and translation, and understanding of how translation systems exploit auxiliary factors to determine the starting point and ending point of messenger RNA translation. These are coupled with models for the ribosome, recently supported by X-ray crystallography, to build a complex picture of synthetic biology in this space.

? DESCRIPTION (provided by applicant): Only a few inexpensive drugs can be used in the developing world for the treatment of AIDS. Several of these target the HIV reverse transcriptase (RT). Accordingly, patient therapy with these drugs often fails when the gene encoding RT undergoes mutation. Thus, the WHO recently reported that after 12 months of treatment with anti-RT drugs, patients most often relapsed when the following mutations arose in RT: (a) for nevirapine, K103N and Y181C; (b) for tenofovir and d4T, K65R; (c) for 3TC and FTC, M184V; and (d) for thymidine analogs, D67N. Therefore, both for surveillance and for immediate patient care, the NIAID and the CDC issued an SBIR solicitation for commercial technology transfer to develop an assay that could quickly and inexpensively detect these five mutations in patient samples in a resource-limited environment. The solicitation sought, as a benchmark specification, a level of detection (LOD) of 10,000 molecules/mL. The work proposed here will deliver this assay with a much better LOD (10-100 molecules/mL), exploiting technology developed under an NIAID R01 to the FfAME that ends in 2014. Thus, an STTR grant format has been chosen to deliver an assay with the following features that make it easy and inexpensive: 1. The assay will do multiplexed amplification for regions of the RT gene that contain resistance-conferring mutations in one assay, avoiding the cost of five separate assays for each of the alleles. This performance specification is possible because of FfAME-Firebird self-avoiding molecular recognition systems (SAMRS). 2. The assay will exploit the isothermal helicase-dependent amplification (HDA), not standard PCR. This avoids both the cost of a PCR instrument and the power demands of thermal cycling. Multiplexed HDA relies on technology recently developed in the Benner laboratory under the NIAID R01 grant, including SAMRS-reverse transcriptase HDA, which has a level of detection (LOD) of 10 ~ 100 molecules. 3. To ensure high coverage, multiple primers covering ~90% of the sequence diversity surrounding the target site will be used. SAMRS, by preventing primer-primer interactions, makes this multiplicity of primers possible, and allows them to be expanded, without needing to redesign the multiplex. 3. The assay will amplify target xNA before SNP detection, exploiting the very low noise of nested PCR using the FfAME-Firebird technology known as artificially expanded genetic information systems (AEGIS). 4. The assay will detect amplicons using orthogonal beacons, also developed here. These also rely on AEGIS technology to suppress background noise, allowing detection by eye of as few as 1010 amplicons. 5. Readout will use immobilized beacons, with fluorescence generated by a hand-held battery-operated LED, with diagnosis made on the spot or, if captured by a cell phone camera, at a remote evaluation center. 6. Should cross-reactivity be observed, in Phase 2, it will be reduced using aminoxy reversible terminators with engineered polymerases, another innovation coming from the Benner laboratory.

? DESCRIPTION (provided by applicant): Technology to deliver molecules on demand that bind to targets or catalyze reactions of a technologist's choosing would have enormous commercial value in research, manufacturing, and medicine. Thus, this has been a holy grail of molecular science for a half century. Even today, chemical theory is inadequate to support de novo design of receptors, ligands, or catalyst having useful affinities or catalytic power. Thus, many have sought to bring Darwinism into the laboratory. Here, the hope is to re-create, under control of a technologist, the evolutionary processes that create the powerful receptors, ligands, and catalysts found in Nature. One approach to create laboratory Darwinism was begun by Gold, Szostak, and others to exploit the intrinsic ability of natural DNA and RNA (collectively xNA) molecules to direct their own replication. These and others developed the technology that we laboratory in vitro evolution (LIVE), which places libraries of xNA molecules under laboratory selection pressure to extract those that bind to targets. Unfortunately, because natural xNA molecules have only four building blocks with few functional groups, they have low information density, ambiguous folding, and little intrinsic functional power, and therefore have seen little commercial use. Using synthetic biology and a second generation artificially expanded genetic information system (AEGIS), FfAME and Firebird scientists have created a fundamental breakthrough in LIVE technology. This involves adding, initially two but potentially up to eight, additional nucleotides to standard xNA. These can carry not only functionality found in natural amino acids, but functionality in addition to that seen in natural proteins, including antibodies, which provide current state-of-the-art technology for creating binding molecules. To lay the grounds for this commercialization proposal, we used AEGIS-LIVE to create AEGIS aptamers for customers who were seeking molecules that bind specifically to breast and liver cancer cells. This grant will fund the next logical step, commercializing AEGIS-LIVE via two overlapping business models: (a) A custom research collaboration model, where Firebird staff provide an AEGIS-LIVE service at a fee to generate receptors, ligands, and catalysts that are chosen by customers and clients. (b) A product generation model, where Firebird uses AEGIS-LIVE to generate receptors, ligands, and catalysts of its own choosing, as commercial products in their own right Phase 1 will perform AEGIS-LIVE that, for the first time, targets a specific defined molecule, glypican 3, a protein that is key to the lethality of many cancers. AEGIS-LIVE will be compared with a standard LIVE without AEGIS, as well as antibodies for the same protein target. AEGIS aptamers will be delivered to our customer, Dr. Chen Liu at University of Florida, to support his liver cancer research. As well as generating AEGIS aptamers having value in their own right, this work will demonstrate the flexibility of AEGIS-LIVE, develop supporting infrastructure, and launch commercialization of technology that NIH referees note is a game changer.

Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites Firebird Biomolecular Sciences LLC University of Florida Steven A. Benner John B. Dame Abstract Eradication of Plasmodium falciparum, the parasite that causes the most malarial deaths, is the only way to finally treat the disease in any specific geographical region. Haiti is one such region, where a small population (11 million), geographical isolation, and special ecology make eradication a real possibility. Thus, the Gates Foundation has just committed $29.4 MM to the CDC Foundation to attempt this eradication. Key to eradication is an assay that identifies human carriers, asymptomatic individuals with active parasite infections. That assay must: (a) Require only a small sample of blood, perhaps 100 µL; (b) Detect very small numbers of living parasites, perhaps as few as 10 parasites per sample; (c) Be as simple to operate as a conventional malarial ?rapid diagnostics test? (RDT); and (d) cost less than $1.00 to run. These specifications are virtually impossible with classical molecular diagnostics. However, Firebird has developed many innovations over the past three years that make such specs possible, including (a) sample prep processes that remove biohazard, (b) whole nucleic acid capture without centrifugation, (c) isothermal nucleic acid amplification using self-avoiding molecular recognition systems (SAMRS) and artifi- cially expanded genetic information systems (AEGIS), and (d) AEGIS molecular beacons. Firebird has shown that these support assays that, for example, detect 22 mosquito-borne RNA viruses in one mosquito carcass. The work is guided by work in the Dame lab showing that P. falciparum ribosomal RNA, present at ~104 copies per parasite, can be detected in malaria-infected blood using RT-PCR. The PCR assay does not meet the cost specs necessary for a LRE, and is not as easy to run as an RDT, but it shows the sensitivity of an assay directed at falciparum rRNA, where detection is robust if a sample of blood contains just one organism. This project will combine these innovations to develop an assay that detects falciparum rRNA as easily as an RDT, but which much higher sensitivity. One strength of the approach is its use of realistic samples of live falciparum in real blood to do benchmarking. Costing less than $1.00, the assay will generate fluorescence if falciparum is present that can be read directly or transmitted by cell phone camera for remote confirmation. We will benchmark (i) a sample preparation work-flow that releases P. falciparum rRNA that is (ii) captured on a solid support, where (iii) captured rRNA is amplified isothermally using primers with SAMRS and AEGIS nucleotides, with (iv) amplicons detected by AEGIS beacons. Results will be compared to samples analyzed by RT-PCR. Metrics for success include an ability to detect 10 parasites in a sample. In Phase 2, the efficacy of the test will be validated in a small field trial with blood from individuals from in a malaria-endemic area of Haiti, again comparing with RT-PCR. This will, as well, support the epidemiological science behind carrier identifica- tion and eradication, as we still do not know the lowest level of parasitemia that can remain stably in an individual. The product should gain WHO and Gates Foundation support, and yield LRE tests for other agents.

? DESCRIPTION (provided by applicant): To many (notably Craig Venter), synthetic biology means simply synthesizing large amounts of DNA. This type of synthetic biology actually began in the 1980's, when Caruthers introduced a phosphoramidite-based solid phase DNA synthesis architecture. This allowed ICI and the Benner group to synthesize complete genes encoding biomedically useful proteins and enzymes for the first time. The second example showed how biotechnological goals could better be met if the synthetic sequences were different from the sequences presented by Nature, through codon optimization and watermarking, inter alia. Subsequent developments now allow semi-routine synthesis of genes; these are used in biotechnology, gene therapy, RNA therapy, and elsewhere. Extrapolation suggests that routine synthesis of whole genomes will soon be possible, hopefully for less than the ca. $30 million spent to synthesize one in the Venter laboratory. Unfortunately, DNA has a rich biophysical chemistry that defeats any architecture that relies on autonomous assembly to make large DNA (L-DNA) constructs by simply mixing synthetic fragments, even within recombinogenic organisms. However, two innovations from the FfAME provide a new approach to creating L-DNA constructs. The first is an artificially expanded genetic information system (AEGIS). AEGIS is a DNA-like molecule that adds eight additional nucleobases that form four additional pairs (the Z:P, S:B, K:X, V:J pairs) to the four natural nucleotides (which form G:C and A:T pairs). By increasing information density of DNA and avoiding non-canonical interactions, AEGIS allows autonomous self-assembly of dozens of fragments to generate L-DNA. The second innovation is transliteration technology. Transliteration allows rule-based replacement of AEGIS nucleotides by standard nucleotides after a L-DNA assembly is complete. By converting S, B, Z, and P to T, A, C, and G (respectively), the AEGIS components can be replaced after they have served their role to guide autonomous self-assembly, converting GACTSBZP L-DNA to entirely standard GACTTACG DNA. To persuade commercial partners to engage with this technology, FfAME scientists demonstrated this strategy using the simpler GACTSB six-nucleotide AEGIS DNA to give, in one assembly step, an active, full-length, and sequence-correct gene encoding kanamycin resistance. Parallel attempts with standard 4-base DNA failed. Phase 1 project will transfer these innovations to Firebird, which will deliver L-DNA and whole plasmids by custom synthesis using an eight-letter GACTSBZP alphabet. This will require (a) adapting OligArch software to support design with this strategy, (b) creating a pipeline to synthesize DNA 60-80 nucleotide fragments using this alphabet, and (c) providing demonstration products, plasmids coding multiple enzymes for complete metabolic pathways to natural products, assembled in a single step. In Phase 2, this technology will be merged with Firebird's E. coli SEGUE strain (Second Example of Genetics Undergoing Evolution) that manages expanded DNA alphabets in cloning vehicles, with one week order-to-clone times for plasmids and viruses.

Commercial Polymerases to Create Receptors, Ligands, and Catalysts on Demand Firebird Biomolecular Sciences LLC University of Texas Steven A. Benner Andrew D. Ellington ABSTRACT Innovators at Firebird Biomolecular Sciences LLC have created several generations of an ?artificially ex- panded genetic information system? (AEGIS) that adds eight nucleobases that form four additional pairs (Z:P, S:B, K:X, V:J) to the four natural nucleotides that form the standard G:C and A:T pairs. AEGIS components carry functional groups not found in natural DNA and RNA (collectively xNA) (-NH2, -SH, -COOH), as well as some not found in any natural biopolymer (e.g. ?B(OH)2 and -ONH2). Thus, AEGIS is a new kind of polymer, replicable, evolvable, and adaptable (like DNA) but also having much of the functional potential of proteins. In DNA, AEGIS has been remarkably successful. Six-letter GACTSB AEGIS DNA enables two FDA-approved products that measure hepatitis and HIV viral loads; here the ability of AEGIS DNA to pair only with AEGIS DNA, and not to background xNA ?orthogonality?, allowed these products to earn ca. $100 million each year in their ten year life. The orthogonality of GACTZP AEGIS DNA, as well as its ability to be replicated with high fidelity by DNA polymerases, are today exploited in diagnostics and surveillance for respiratory disease viruses (common and exotic), arboviruses (in mosquitoes), noroviruses, and coronaviruses (e.g. SARS, MERS). Firebird now sees the opportunity to expand its product portfolio from AEGIS DNA to include AEGIS RNA. Its scientists recently reported large-scale syntheses of AEGIS RNA triphosphates and phosphoramidites; these are now sold in its 2016 catalog. Further, Firebird scientists have shown that T7 RNA polymerase makes AEGIS RNA from AEGIS DNA, although inefficiently, and found reverse transcriptases that make AEGIS DNA from AEGIS RNA, in both six and eight letter versions (e.g. GACTSBZP). These are ?leads? for the proposed research. RNA is well-known to have a richer set of folds and more intrinsic potential to serve as receptors and ligands (aptamers, a term and technology invented by the subcontractor). Functionalized AEGIS RNA will thus likely be better than AEGIS DNA in these biomolecules. Functionalized AEGIS RNA will also have enhanced value in laboratory in vitro evolution (LIVE) experiments to create receptors, ligands, and catalysts on demand. Should this vision be realized, it will create a revolutionary transformation of diagnostics and therapeutics. This vision requires better RNA polymerases and reverse transcriptases. While Firebird could undertake their development in-house, advances in the Ellington laboratory at the University of Texas suggests that collabora- tion would achieve this goal faster. Ellington has already created directed evolution platforms to give T7 RNA polymerases and thermostable reverse transcriptases that accept unnatural (but not AEGIS) nucleotides. This collaboration will apply those platforms to create enzymes that interconvert AEGIS DNA and AEGIS RNA. The enzymes themselves will be commercial products; they will also, however, support LIVE with this evolvable functionalized AEGIS biopolymer. Phase 1 will show the feasibility of using Ellington?s platforms to create these enzymes. Given feasibility, the platforms will be applied in Phase 2 to complete 12 letter AEGIS alphabets.

No single advance at the frontier between biology and chemistry will transform research in the life sciences more than the synthesis of an artificial life form (a ?xenobiotic?). This xenobiotic will reproduces much of what we value in natural life (including its ability to grow, evolve and adapt), but with a different core molecular biology. This would move beyond the visions of ?synthetic biology? and ?biomimetic chemistry?, the second recognized just this week with a Nobel Prize in chemistry, to deliver an artificial biology, a new field that will transform both biomedical science and technology for decades to come. This goal transcends the current orientation of the NIH towards ?descriptive biology?, a research paradigm that is already well represented in the Director?s portfolio. Indeed, we begin by assuming that those now supported by the Director in these now-classical descriptive strategies will eventually complete this research programme, producing robust pictures of biomolecular structure from the atomic scale to the macroscopic scale. The work proposed here will lay the grounds for the life sciences that will follow next. A paradox is built into any application to do transformative research. If it is truly transformative, one cannot anticipate the details of its impact. This is problematic for peer review. To manage this paradox, we list a half- dozen technological capabilities that our artificial life form should deliver based on what in vitro preliminary work has already delivered. These include transforming the way we generate clinically used receptors, ligands, and catalysts, lowering the cost of diagnostic kit production, and creating new classes of engineered proteins. An analogous paradox arises respect to the science. Many now argue that the touchstone for ?understanding? must be the ability to design and synthesize. However, we recognize that theory (now, and far into the future) is inadequate to design without support from Darwinism. Our global strategy therefore combines design and Darwinism. This strategy for discovery and paradigm change cannot be matched by hypothesis based research. Again, discovery and paradigm change cannot be predicted, creating another peer review issue. We manage this by examples from in vitro studies, which have transformed our understanding of nucleic acids. Consequently, this proposal contains an unexpectedly large number of preliminary results. This places it at risk of being excluded from a transformative research program under a hope can its transformation can funded as a standard NIH project. This hope has been fully shown to be quixotic; the experiment has been repeatedly tried and failed, in part because of the high risk still associated with efforts to achieve the first needed breakthrough. To manage this risk, the program is structured to give many routes to success. Balancing this is the unarguable fact that if any one of the routes to success is traversed, the result (an artificial Darwinian life form) will be as memorable as any of the many accomplishments in the history of biological-chemical science. Last, we provide a detailed discussion of biosafety with respect to artificial life.

Nucleic Acid Innovations to Manage Pathogen Sequence Divergence Foundation for Applied Molecular Evolution Steven A. Benner Zunyi Yang ABSTRACT One of the most important outcomes from synthetic biology has been the recognition that many limitations of diagnostics tools that target the DNA and RNA (collectively xNA) arise from defects in the xNA molecular framework. These defects can be fixed by changing the structure of DNA used in these assays. The Benner lab has created over a dozen reagent, enzyme, and architecture innovations based on this recognition. These are now allowing assays to move from the clinical lab to emergency rooms to physicians offices. In this progression, one problem arising from the molecular biology of the RNA viruses has remained recalci- trant. The sequences of RNA viral genomes diverge rapidly. Thus, we do not know the exact sequence of the xNA molecules that we are trying to detect in any patient. With increasing frequency, this allows RNA viruses to escape primers and probes needed to detect their xNA. This, in turn, renders simple tests unable to detect a virus with certainty. When these are intended to diagnose individual patients, this all but prevents FDA approval under anything but emergency use authorizations. Further, this uncertainty in the target xNA makes medically informative variation difficult to detect amid the medically uninformative variation. This work will introduce NextGen ?biversal? nucleic acid innovations to manage this problem. We begin with by noting that viral sequence divergence occurs under strong adaptive constraints with error biases intrinsic in relevant polymerases. Thus, transitions (purines replace purines, pyrimidines replace pyrimidines) are more common than transversions (purines replace pyrimidines, or vice versa). This means that we can manage viral genome divergence with pyrimidine biversals (Y) that pair to both A and G, and purine biversals (R) that pair to both C and T. Biversals have advantages over ?universal bases?, which create primers so promiscuous that they get lost by binding to background xNA, abundant in real biological samples. These facts set up these aims. Aim 1. Add NextGen biversals to isothermal amplification architectures, which allow diagnostics kits to move closer to points-of-sampling and points-of-care. We will benchmark their performance relative to the performance of amplifications with all-standard primers, and quantiate specificity footprints. Aim 2. Characterize the details of how polymerases handle NextGen biversals, specifically, what standard nucleotides are placed opposite these biversals by polymerases used in low-temperature amplifications. Aim 3. Integrate NextGen biversals into architectures for SNP detection that exploit ligation and cleavage. Rich preliminary data allow this project to move directly to a development stage. The emergence of Zika, Ebola, chikungunya, dengue, and other RNA viral pathogens, and the soon-to-emerge Mayoro virus, points to the immediate significance of this project. Especially innovative in this project is its integrated investigation of alternative molecular structures, the enzymes called upon to handle them, and the architectures where they will be used. These will allow FDA approvable diagnostics products to move towards points of sampling.

Urgent Needs in Surveillance and Diagnostics. Zika Firebird Biomolecular Sciences LLC Florida Medical Entomology Laboratory, the University of Florida Steven A. Benner Barry W. Alto ABSTRACT The potential hazards of the Zika virus to both American citizenry and citizens of the world need no discussion. We are still learning the distribution and impact of the virus on human development in utero, as well as its distribution in patient saliva, sexual fluids, and urine. With several dozen cases in the US at the time of this writing, most obtained overseas, the extramural research programs of public health agencies are being asked to seek better public health surveillance technologies and immediate human diagnostics tools for Zika. For example, in Brazil, the immediate need is a test that to distinguish between dengue, chikungunya, and Zika in public clinics; at this time, this distinction is made based on interrogation of patients with respect to the timing of the appearance of various symptoms. The work proposed here will deliver those technologies and tools for Zika. However, this work will provide more. Today, the NIAID, CDC, and other public health units responsible for managing infectious disease outbreaks move from disease to disease, with each new outbreak and news cycle. This is inefficient, at best. Efficient public health services in an age of emerg- ing diseases need a robust and reproducible technology platform that is sufficiently flexible to sur- vey for any emerging infectious diseases without crisis. The work proposed here offers such a platform as part of its ability to survey for Zika, and distinguish it from other arboviruses, based on reagent innovations drawn from the field of ?synthetic biology?, including artificially expanded genetic infor- mation systems (AEGIS), self-avoiding molecular recognition systems (SAMRS), biversal nucleotides, transliteration, and evolution-based arbovirus sequence analysis, in a collaboration between the Flor- ida Medical Entomology Laboratory (FMEL), which has live lab-infected mosquitoes with Zika, dengue, and chikungunya, and Firebird, a serial innovator in synthetic biology and diagnostics technology. This R21 project will first add Zika to an already established kit that detects 22 other arboviruses, common and exotic.13 We will benchmark that expanded kit, delivering it to beta testers in FMEL, where multiplexed readout is done using a Luminex instrument. This independent testing guarantees scientific rigor, and will be done by Month 12. In Year 2, we will seek a simpler system, focusing the multiplex on geography-specific target sets, adding biversals to manage sequence evolution of the RNA viruses, replacing Luminex in 10x target assays by a readout based on a 4x4 micro array, and going as far as possible towards a 10x multiplex system that can assay ~ 10 mosquitoes in the field. Towards these ends, we will meet the following specific aims: The outcome of this project will include new Zika science, as well as kits that will allow public health services to enjoy the advantages of these reagent innovations in managing the Zika outbreak, just as those advantages are now enjoyed in many products, including FDA-approved products, for human diagnostics.

Point-of-sampling detection of Lyme and other tick-borne diseases Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT Lyme disease is today the most prevalent vector-borne disease in the US, and ticks in general are the most significant vectors for human pathogens worldwide, including Borrelia, Babesia, and Rickettsia species. For Lyme disease, the CDC confirmed only 28,000 cases in a population that might actually hold 300,000. This severe underdiagnosis is due to: (a) expensive and spotty environmental surveillance, yielding insufficient patient and physician awareness, and (b) inadequate molecular tests; those exploiting antibodies can give 86% false negatives. Further, to order a molecular test (at $260), a physician must guess that a patient suffers from one of these diseases. As single pathogen-single tests are the norm in the FDA-regulated diagnostics space, these guesses must expand (each $200) to other candidate pathogens, one at a time. Not surprisingly, aggressive advocacy groups have arisen to counter (what they feel to be) inadequate CDC/NIH response to this disease threat. This R21 project will deliver a molecular test that, in one step in the hands of uncertified users, identifies a complete panel of all tick-borne pathogens, for less than $1.00, and within 30 minutes. The assay will apply to ticks directly, either trapped or pulled off a pet or a child. It will be useable by college nurses, doctors in their offices, public health service staff, and even laypeople, to examine ticks. After this R21 project is complete, the data will set the stage for a human diagnostic kit that can be FDA-approved to apply to patient samples. Although the Benner lab has never before worked with tick-borne diseases, this proposal has a high chance for success, as it has been de-risked by work, just published, where the lab delivered a kit that detects dengue, chikungunya, and/or Zika in a single assay, in 30 minutes, in a single mosquito carcass. The sample is rendered free of biohazard by treatment with ammonia (similar to Windex®) and ethanol, and dropped into a pre- prepared tube containing dry reagents, shipped without refrigeration. Alternatively, urine or blood may be used. This kit is being used today to monitor the presence of pathogens in trapped mosquitoes, and shipped to China and India, where it detects dengue and chikungunya in human urine and blood. This illustrates our strategy of developing unregulated tests to support introduction of tests for patient use. The deliverable will be benchmarked on real ticks containing real pathogens, provided by the laboratory of Maria Diuk-Wasser at Columbia. This will include blind testing on ca. 50 samples. The premises are: (a) The NH3-EtOH disruption is sufficient to release detectable amounts of pathogen target; (b) multiplexing in a reverse-LAMP architecture can be increased from 3 to ~10 to cover all tick targets; and (c) AEGIS in displace- able probes will support a patterned readout for different pathogens, and a control. The authentication of the reagents will be assured by making the deliverable available to the public, as is being done for mosquito- borne pathogen detection.

Point-of-sampling detection of Human Papillomavirus (HPV) Foundation for Applied Molecular Evolution Steven A. Benner Ozlem Yaren Human papillomavirus (HPV) infection is associated with cervical and oral cancers. Early detection with one of over 100 molecular tests, costing $100-200 each, is responsible for a substantial decrease in HPV-caused death in the developed world. Public health communities are eager to see this success mirrored in the developing world. More widespread screening will further slow the spread of this STD, and further reduce fatality from it, if the assays are less expensive, return results in minutes rather than weeks, and yield greater patient compliance among men and women. This project will deliver the next generation of HPV diagnostics to move the test down the food chain towards rapid (<60 min) calling of results at points-of-sampling, in doctors' offices, and in rural areas lacking doctors. The last is especially important in developing countries, where cost is a big constraint. This rules out polymerase chain reaction (PCR) to amplify HPV DNA in favor of isothermal amplifications. The goal of this R21 is to assess the ability of an innovative variant of the loop amplification (LAMP) isothermal amplification architecture, artificially expanded genetic information systems (AEGIS), and other innovations in nucleic acid chemistry, to deliver this product. Here, development of a point-of-sampling kit to detect HPV variants will go hand-in-hand with its use in a truly low resource environment, a reference laboratory run (at no cost to this grant) by Prof. Dr. Xian-Ping Ding at the Sichuan University in China. His laboratory will use fully protected patient samples to compare results from our kit with gold standard kits, and return results with complaints and suggestions. Development work here allows this project to proceed on an The Aims are to a large extent de-risked by R21 budget, presses us to have realistic costs, and gives us access to rural areas that are truly low resource, in a sense that exists nowhere in the US. work just published, where the Benner lab created a kit that detects dengue, chikungunya, and/or Zika in a single assay, after 30 minutes, in a single mosquito carcass, captured on Q-paper. The sample is rendered free of biohazard by treatment with ammonia and ethanol, and then dropped in a pre-prepared tube containing lyophilized reagents; these are shipped without refrigeration. Alternatively, urine or plasma is sampled (one drop). After being heated at 65 °C, the assay results are viewed by reading through fluorescence output generated by blue LEDs mounted in a $10 device. The assay itself costs less than a dollar, has <1% false positives and <1% false negatives, and is presently being manufactured under a contract in Florida for environmental surveillance. The final deliverable will be a statement about the feasibility of the kit, useable by individuals who are trained in its use but not otherwise credentialed or licensed, to, within 30 minutes, tell if a mucosal swab contains one of two high risk HPV variants, and one or more low risk HPV strains. 1

Create Ultralong DNA Constructs in One Assembly Step Firebird Biomolecular Sciences LLC Steven A. Benner Foundation for Applied Molecular Evolution Shuichi Hoshika Abstract Frost & Sullivan found a 2014 global market for DNA oligos at $241 million, $137 million for genes. Private investment in DNA synthesis companies like Twist, Ginkgo, and DNA Script give collective valuations of several billion dollars. Federal public investment by the NIH, DARPA, and others in synthetic biology that depends on DNA synthesis exceeds $100 million annually. These numbers stand behind this project to develop two innovations to (a) deliver, under a custom synthesis model, long DNA (L-DNA) assemblies (b) secure a licensing platform, and (c) create collaboration and buyout opportunities. These technologies are: 1. Artificially expanded genetic information systems (AEGIS), which add 4 nucleotides forming 2 additional orthogonally binding nucleobase pairs (Z:P and S:B) to the pairs (C:G and T:A) found in natural DNA. Eight- letter DNA increases the number of sequence accurate fragments that can be autonomously assembled. 2. Transliteration, which converts Z, P, S and B to C, G, T and A respectively, giving an entirely natural L- DNA construct by removing the AEGIS components after they have done their job assembling fragments. Highlights of Phase I results include: (a) OLIGARCHTM software predicting stability of 8-letter GACTZPSB DNA duplexes. (b) Fidelity of DNA products made by AEGIS + transliteration as good as in commercial G-blocks. (c) Constructed genes for kanamycin resistance and green fluorescence protein were active in E. coli cells. These successes shift the cost/quality burden for L-DNA synthesis towards residual error management. Aim 1. Manage residual error using, as experiments suggest: 1.1 C-glycosides to eliminate depurination and depyrimidinylation, should these cause residual error. 1.2 Enzymatically removable protecting groups to eliminate chemical damage during deprotection. 1.3. Capturable capping groups to achieve simple >99.999% removal of truncated species. 1.4 Enzymatic DNA synthesis to eliminate all chemical degradation in harsh phosphoramidite synthesis. Residual error will be further managed using MutS and Surveyor nuclease error correction. Aim 2. Create synthetic pipelines to prepare the building blocks and reagents used to manage residual error. Aim 3. Develop array-based phosphoramidite synthesis of fragments with continuous error evaluation. Reproducibility will be ensured by making the reagents themselves available for sale. This is a source of immediate revenue as well as a major part of our marketing strategy. Already, reagent sales to satisfied customers have yielded licensing deals worth over $2.5 MM. For commercialization, Firebird just executed an agreement with DNA Script, a pioneer for non-templated enzymatic DNA synthesis and its automation, to go forward after Phase 2, should enzyme-based DNA synthesis be preferred to manage residual error. This includes licensing Firebird's patents for enzymatic cyclic reversibly terminated untemplated DNA synthesis.

Field Detection of arboviruses directly from mosquito traps Firebird Biomolecular Sciences, LLC. Abstract Mosquitoes are the world's deadliest creatures. In the US, mosquitoes are vectors for West Nile, many kinds of encephalitis, and viruses arriving Latin America and Africa. In the developing world, they cause millions of cases of disease and death each year. Routine insect spraying is not a fix, as it leads to pesticide resistance, encounters public opposition, and destroys beneficial insects. Thus, the EPA, CDC, ECDPC, and their counterparts around the globe, strongly recommend surveillance before spraying. Florida law requires it. In Phase 1, we created (a) innovative technology to generate CO2 to attract mosquitoes, (b) innovative tech- nology to collect viral RNA from the attracted mosquitoes, (c) data showing that collected RNA is stable under Florida summer conditions for at least 3 days, (d) processes where captured viral RNA is amplified directly without handling mosquitos or Qiagen RNA preps, and (e) reagents that allow robust multiplex amplification. These specs, which can transform the science and practice of arbovirus surveillance, attracted TrakITNow, an innovator in instrument design. Its award-winning trap (Moskeet) counts and speciates mosquitoes, doing everything short of collecting samples from the mosquitoes and determining if these hold arboviruses. This Phase 2 project will re-configure and metric the Phase 1 innovations to allow them, in Phase 3, to be placed on Moskeet in two operational architectures (OA1 and OA2). A series of improvement and adaptations will be evaluated using tightly crafted metrics in two Aims. Meeting these metrica will trigger a commitment by TrakITNow to support Phase 3 funding to launch a combined product, Aim 1 will develop and metric Firebird's chemistry for an OA1 architecture that will convert Moskeet from a trap-count-speciate-communicate product into a trap-count-speciate-sample-communicate product. This will exploit Firebird's Phase 1 innovation (SterileQTM) that autonomously traps and stabilizes viral RNA. Different baits, RNA-stabilizing preservatives, and pre-amplification enzymes will be metricked to create, at the end of Year 1, a Moskeet-ready OA1 chemistry that allows continuous sampling of mosquitoes. Metrics include (a) >50% of infected mosquitoes leaving detectable sample, (b) >200 hours half-life of RNA, and (c) capture of >1 pg of RNA. An instrument from Insilixa (Hydra), also enhanced by Firebird's reagents, will be used for on-site virus detection. Hydra is portable, and its low complexity design allows it to be used by amateurs. Aim 2 will develop and metric Firebird's chemistry for an OA2 architecture. This will convert Moskeet into a trap-count-speciate-sample-analyze-communicate product. Here, we will develop and benchmark point-of- sampling amplification tools, specifically: (a) RT-PCR, and (b) loop amplification (LAMP). These will, as needed, incorporate reagent innovations (AEGIS, SAMRS, Biversals) to support highly multiplexed detection of arboviruses, create uniform amplification of multiple analytes, manage primer-primer products, prevent loss of PCR resources to off-target processes that create monsters, and manage arbovirus sequence divergence.

Basic Research to Diagnostics and Surveillance in Lower Resource Environments Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT We will deliver to the NIAID and CDC communities, through basic research, a scientific understanding of pairing, mispairing, and enzymology of natural DNA and RNA (collectively xNA) that goes deeper than the axiom that A pairs with T, and G pairs with C. The experiments are designed to learn: (a) Why robust multiplexed PCR (mPCR) for clinical use seems impossible with more than 20-30 targets. (b) Why conventional expedients (including careful primer and probe design, internal nesting, and external tagging) fail to robustly support multiplexing beyond ~30 targets. (c) Why those failures are not reproducible from sample to sample. (d) Why conventional multiplexes targeting n targets often collapse when an n+1th target is added. This prevents, when a new pathogen emerges (as for 2019-nCoV), a diagnostics maker from simply adding a new target to an existing mPCR kit, thereby meeting the emergency need. (e) Why manufacturing specs become increasingly more demanding as the level of multiplexing increases. These problems restrain 21st century diagnostics to two 20th century design and regulatory paradigms. (i) A guess-then-test paradigm for singleplexed molecular diagnosis, which requires physician to guess which pathogen might be associated with patient malaise, prescribe a ~$150 singleplexed test based on that guess, and re-prescribe further tests until a guess proves correct. (ii) The inflexible-multiplexed-panel paradigm. Here, assays are bundled into a multiplex appropriate for a specific sample and symptom set; failure (d) prevents that multiplex from changing for emerging diseases. By developing the science of both natural and unnatural DNA (including artificially expanded genetic information systems, AEGIS, and self avoiding molecular recognition systems, SAMRS), this project will deliver to researchers, manufacturers, and the FDA science to meet the 21st century NIAID mission. We will: Task 1. Complete thermodynamic and enzyme rules to place SAMRS optimally in primers that target both DNA and RNA. Rules will be metricked by comparing predictions made with these rules to experiments. Task 2. Metric, by deep sequencing, mPCR failures (a) through (e). Task 3. Metric how AEGIS and SAMRS mitigate or eliminate failures (a) through (e). Task 4. Identify failure modes that arise with RNA targets specifically. Since RNA has folding options not available to DNA, these modes may be especially resistant to nucleic acid innovations. Task 5. Build a body of statistical knowledge for AEGIS-SAMRS mPCR, especially with respect to add-ons, quantitative amplification, and manufacturing tolerances. This will help move away from guess-then- test and inflexible-multiplexed-panel paradigms, lowing cost, supporting FDA regulatory processes, and better managing pandemics. 1

Enzymatic Synthesis of RNA Foundation for Applied Molecular Evolution Thomas Jefferson University Steven Benner Richard Pomerantz ABSTRACT The demand for synthetic RNA in biotechnology, research, and the clinic has increased dramatically in the last few years. This is due inter alia to novel CRISPR-Cas9 genome engineering techniques, the re-invention of aptamers and aptazymes with picomolar affinities using expanded genetic alphabets, and investigations of small RNAs in mammalian biology, all relying on synthetic RNA. Even with some of the best firms advancing classical phosphoramidite chemistry, 20 nmoles of an 120 nucleotide Ultramer® still costs $1080, a severe limit on researchers asking Why not? and What if? questions using synthetic RNA. The cost of RNA would be dramatically lowered if phosphoramidite chemistry were replaced by enzyme- assisted RNA synthesis. Two advances make it now timely to achieve this Grand Challenge. Chemistry. The Benner lab invented a removable 3'-O aminoxy (ONH2) group for NextGen sequencing. Now licensed to DNA Script in a dual use mode for enzyme-assisted DNA synthesis, aminoxies generate 200- mers in good purity and yield. In a virtuous cycle, this led us to develop low cost solid-phase methods to make aminoxy triphosphates at < $1/micromole, and methods to make 3'-O-aminoxy ribonucleoside triphosphates. Enzymology. Marc Delarue (collaboration letter), DNA Script (collaboration letter), and Richard Pomerantz (co-Investigator) discovered enzymes, including polymerase ? and its variants, that add ribonucleosides to an RNA primer. This creates an architecture for enzyme-assisted RNA synthesis based on aminoxy termination that complements a classical architecture that exploits RNA ligase. In Aim 1, we will use a classical architecture involving the ligation of nucleoside 3',5'-bisphosphates to learn how to manage folding that occurs in natural RNA during enzyme-assisted synthesis. Even more than with enzyme-assisted DNA synthesis, this folding obstructs the synthesis of a full range of RNA sequences. Novel transformable, self-deprotecting, and soft deprotectable modifications should allow this problem to be resolve. As Aim 2, we will engineer Pol ? variants to find those that accept the 4 standard nucleotides in a Fig. 4.2 architecture that exploits 3'-ONH2 reversible terminators. These will be metricked by (i) rate of incorporation, (ii) sequence independence of incorporation, and (iii) length dependence of these. The principal sources of error (coupling failure leading to single nucleotide deletion) will be rigorously metricked As Aim 3, we will implement a semi-automatic platform for RNA synthesis. We will also use ligases and Pol ? variants to incorporate next generation nucleotide analogs that have value in therapeutic RNA, RNA aptamers and aptazymes, and RNA tagging. This will attract commercial instrument makers (e.g. DNA Script and Nuclera were both contacted about this platform) to adapt their instrument to our chemistry/ enzymology. Even before this happens, our semi-automatic platform will allow this technology to be transferred to NHGRI centers that are chosen under NHGRI RFA-HG-20-019, a parallel RFA now accepting applications.

Easily Used Kits to Evolve Reagents that Covalently Tag and Inactivate Proteins Foundation for Applied Molecular Evolution Steven Benner ABSTRACT Under PAR-19-253, the NIGMS seeks new technologies that create a positive feedback loop that drives science forward by allowing new questions to be asked and new discoveries to be made, which in turn drives the development of new technologies. The Benner group has, for 30 years, contributed to this NIGMS vision, developing new technologies for NextGen DNA sequencing and NextGen DNA synthesis, bioinformatic and evolutionary analyses, dynamic combinatorial chemistry for drug discovery, protein engineering, and new platforms that make multiplexed diagnostics easy, platforms used today to manage the COVID pandemic. Here, we offer the NIGMS another transformative tool, a platform to allow researchers to choose a protein target and create a reagent (an AEGISZyme) that chemically transforms bound proteins. Such reagents have been sought for 40 years with only limited success. We will focus on one transformation: AEGISZymes that add an acyl group to an amino group on a lysine of the bound target, where the acylation reagent is an ester. This acylation may inactivate the targeted protein, allow- ing researchers to test hypotheses about the role of that protein in biology. It may carry a payload which, when internalized with the target protein, carry drugs or stabilized AEGISZymes into a cell. It may fluorescently tag the protein to help clinicians cut away fluorescing cancer cells selectively as they resect a tumor. To achieve this transformative and innovative outcome, we will apply laboratory in vitro evolution (LIVE) to artificially expanded genetic information systems (AEGIS). The platform will be delivered by meeting 3 Aims: Aim 1. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines in target proteins with pass/fail reaction times of <10 sec-1. To test this, we will create these AEGISZymes that use a co-substrate carrying an ester group for three targets. Rates of the selected AEGISZymes will be quantitated, specificity will be metricked against similar targets with slightly different amino acid sequences, and modification sites will be found, Aim 2. We will use AEGIS-LIVE to deliver AEGISZymes that acylate lysines on researcher-chosen targets with turnover, with pass/fail turnovers of >1000 and kcat/KM of >105 M-1 sec-1. Turnover rates will be metricked under physiological and laboratory conditions, and correlated to duplex stability from thermodynamic data. Aim 3. We will use AEGIS-LIVE to deliver mirror AEGISZymes that are stable in biological systems, including transport into cells. This will allow AEGISZymes to be used in biological media, to support nanotrain toxin delivery, and to set the stage to use these molecules in vivo. Aim 4. We will test the scope of the platform to address design parameters, such as how long random regions should be, how good loops are as full protein surrogates, and how sequence space is searched. Last, to lay to rest any view that AEGIS-LIVE is too cumbersome, we will create distributable kits that allow their recipients to make their own AEGISZymes. This is the ultimate in authentication and reproducibility.

Summer Research Administrative Supplement to 1R01GM128186-01 Transforming Life Sciences: Artificial Life Foundation for Applied Molecular Evolution Steven A. Benner ABSTRACT In addition to its research, teaching, and outreach activities, the Foundation for Applied Molecular Evolution runs a program that offers opportunities for laboratory research experience to undergraduates seeking careers in medicine, biotechnology, and biomedical research. In the past few years, undergraduate trainees have gone on to MD-PhD programs (Michael Durante, Oleg Uryasev, Jennifer Le), to start biotech enterprises (Heshan Illangkoon), and to pursue Ph.D. degrees (Marc Neveu). All published papers from their undergraduate research. Here, we seek an administrative supplement for summer 2021 to support Justin Kim, an undergraduate from the University of Florida. The supplement will provide him his first experience in protein engineering in an area (DNA polymerases) that has applications not only to the project that this proposal supplements, but also can be applied in diagnostics, in the generation of therapeutic agents, and in biotechnology. The project that he will join (1R01GM128186-01) is a Director's Transformative project that seeks to create an artificial life form (a ?xenobiotic? known as SEGUE). This xenobiotic will reproduce much of what we value in natural life but with a different core molecular biology. This is moving beyond ?synthetic biology? and ?biomimetic chemistry?. Specifically, SEGUE restructures the DNA of the living cell by adapting Watson-Crick pairing. In 4-letter standard DNA, pairing follows two complementarity rules: (a) size (large purines pair with small pyrimidines) and (b) hydrogen bonding (on purine pu and pyrimidine py ring analogs, hydrogen bond acceptors, A, pair with donors D). Rearranging D and A groups on the bases creates artificially expanded genetic information systems (AEGIS). The parent project involves creating new cells that replicate six-letter DNA, either GACTZP DNA or GACTKX DNA. This is done by recruiting E. coli as a platform, and expanding its metabolic capabilities to support six-letter replication. Replication of 6-letter DNA is done by DNA polymerases. This project requires the engineering of these polymerases to accept unnatural nucleotides, this project has led to the publication of many of these polymerases. Many of them have been thermally stable, however. While these work well in PCR, they do not perform as well at low temperature where E. coli lives. At the same time, various reverse transcriptases have been recruited that do accept six letter genetic alphabets. Thus, this background lends itself to a single Aim: Mr. Kim will use protein engineering on a diversity science platform to improve mesophilic DNA polymerases that copy DNA built from six nucleotides. These polymerases will be used in cells being engineered to manage and use expanded DNA, to be an integral part of the funded Director's Transformative Technology award.

Reagents to Chemically Tag Specific Coronavirus Spike Proteins Firebird Biomolecular Sciences, LLC. Steven A. Benner Bharat Gawande Abstract While vaccines and antivirals may be long term solutions to the current coronavirus pandemic, it is now widely appreciated that the pandemic can be managed before these emerge by rapid, point-of-sampling public space entry tests (PSETs), given appropriate FDA regulatory relief. PSETs will be even more important if a good vaccine never emerges, a possibility given experience with other coronaviruses. In the 2003 SARS crisis, we delivered a best in class PCR kit for SARS that targeted coronaviral RNA. This was possible due to our first generation nucleic acid innovations, which were also used in respiratory pathogen panels and cystic fibrosis tests, inter alia. Luminex acquired EraGen in 2011 for $34 million. Since then, the PI generated 2nd and 3rd generation innovations that supported tests for MERS, arboviruses, norovirus and, this year, CoV-19 itself. These include a PSET that gives 10 minute results using proprietary displaceable probe loop amplification. Intrinsic chemistry suggests, however, this is the fastest any RNA-targeted test can be. Here, we will use our 3rd-generation DNA innovations not to target coronaviral RNA, but the coronavirus itself. Firebird will create reagents (AEGISZymes) from an artificially expanded genetic information system (AEGIS) that catalyze covalent acylation of lysines on the surface of ~300 spike proteins per virus. Each acylation will generate ~300 redox active moieties on an electrode surface per virion. The test is conceptually advanced over antibody tests, as it uses stable covalent bond formation, rather than non-covalent antibody: antigen interactions, and exploits electrochemical detection rather than a lateral flow. Our Phase 2 partner, MightyGate, has shown with standard aptamers detection in less than a minute with such architectures. With >100x sensitivity over Abbott BinaxNOWTM, the assay should detect virus in any contagious individual at entrances to arenas, schools, and other spaces as fast has handbags and are checked. Specifically, we will: Aim 1. Under IRB 2020001, we will use AEGIS-LIVE to evolve AEGISZymes in saliva that acylate lysines in spike proteins and peptide loops of CoV-19, SARS, and MERS. Aim 2. Metric the AEGISZymes that emerge, measuring their binding affinities, acylation rates, and specificity among the 3 homologous surface loops. The metrics are: M1. Acylation with kcat ? 1 sec-1. This should be readily achieved based on pmolar affinities that are now routine with AEGIS-LIVE; similar rate constants are now routine in analogous catalysts. M2. Specificity with kcat/Kdiss ratios >103, to ensure that CoV-19 is distinguished from other coronaviruses. If the metrics are met, MightyGate has committed itself to participate in Phase 2 work that will incorporate AEGISZymes onto its kiosk-style instrument. The kiosk has been proven with standard aptamers to give electrochemical readouts in less than 1 minute. Firebird's AEGISZymes will immediately improve the sensitivity of that kiosk, and immediate commercial value. However, our approach is general to any virus target.