Foundation For Applied Molecular Evolution Inc

TRANSFORMING LIFE SCIENCES: ARTIFICIAL LIFE

BASIC RESEARCH FOR DIAGNOSTICS AND SURVEILLANCE IN LOWER RESOURCE ENVIRONMENTS - 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

APTAMERS FROM ARTIFICIAL GENETIC SYSTEMS

CHEMISTRY TO IMPROVE HUMAN GENOMIC ARRAY ANALYSIS ARCHITECTURES

ENZYMATIC SYNTHESIS OF RNA - 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.

SYNTHETIC SUBSTITUTES FOR DNA

DARWINISM FROM ARTIFICIAL GENOMES

NEAR TERM DEVELOPMENT OF REAGENTS AND ENZYMES FOR GENOME SEQUENCING

EQUIPMENT SUPPLEMENT TO 1R01GM141391-01A1 (EASILY USED KITS TO EVOLVE REAGENTS THAT COVALENTLY TAG AND INACTIVATE PROTEINS) - EQUIPMENT SUPPLEMENT FOR 1R01GM141391-01A1 (EASILY USED KITS TO EVOLVE REAGENTS THAT COVALENTLY TAG AND INACTIVATE PROTEINS) FOUNDATION FOR APPLIED MOLECULAR EVOLUTION STEVEN BENNER ABSTRACT THE FOUNDATION FOR APPLIED MOLECULAR EVOLUTION (FFAME) HOLDS A CURRENT NIGMS R01 RESEARCH AWARD (R01GM141391, EASILY USED KITS TO EVOLVE REAGENTS THAT COVALENTLY TAG AND INACTIVATE PROTEINS). UNDER THIS AWARD, FFAME IS REQUESTING SUPPLEMENTAL EQUIPMENT FUNDS THAT WILL BE USED TO REPLACE A 16-YEAR-OLD BIORAD PMI PHOSPHORIMAGER, AN INSTRUMENT THAT SERVES AS THE WORKHORSE FOR THE R01 PROJECT. THE MAIN GOAL OF THE FUNDED R01 GRANT IS TO UTILIZE FFAME’S ARTIFICIALLY EXPANDED GENETIC ALPHABET (AEGIS) TO DEVELOP A PLATFORM TO CREATE RECEPTORS, LIGANDS, AND CATALYSTS ON DEMAND. THESE MOLECULES CAN BE USED TO COVALENTLY MODIFY, TAG, AND DESTROY PROTEINS THAT ARE CHOSEN BY RESEARCHERS AND CLINICIANS TO TARGET AND SOLVE MANY SCIENTIFIC, DIAGNOSTICS, AND DISEASE MANAGEMENT PROBLEMS. THIS ADMINISTRATIVE SUPPLEMENT, IF GRANTED, WILL FACILITATE THE PURCHASE OF AN AMERSHAM TYPHOON BIOMOLECULAR IMAGER, WHICH WILL REPLACE THE BIORAD PMI AND WILL SERVE A CRITICAL ROLE IN THE SUCCESS OF THE FUNDED R01 PROJECT.

BIOCHEMICAL AND PHARMACOLOGICAL STUDIES OF HUMAN MEMBRANE PROGESTERONE RECEPTORS

ALCOHOL METABOLISM,PRIMATE EVOLUTION AND PALEOGENETICS. AN INCLUSIVE PARADIGM

COLLABORATIVE RESEARCH: SYNTHETIC AND SYSTEMS BIOLOGY APPROACHES TO SEMI-SYNTHETIC CELLS WITH EXPANDED DNA ALPHABETS

EXPANDED DNA, IN VITRO SELECTION, APTAMERS, AND CANCER

IMPROVED HIV ASSAYS BY COMBINING FOUR INNOVATIONS IN NUCLEIC ACID CHEMISTRY

IN THE DECADE SINCE HE ARGUED THAT CHEMICAL INSTABILITY PRECLUDED THE USE OF RIBOSE OR ANY OTHER CARBOHYDRATE AS A COMPONENT OF ANY PREBIOTIC GENETIC

ARTIFICIAL GENETIC SYSTEMS

AUTONOMOUS DIAGNOSTICS TO ENABLE PREVENTION AND THERAPEUTICS: DIAGNOSTICS ON DEMAND - POINT OF CARE

"PREBIOTIC ROUTES TO CARBOHYDRATES INVOLVING BORATE MINERALS"AN EMERGING CONSENSUS BELIEVES THAT AN EARLY FORM OF LIFE ON EARTH USED RNA AS ITS ONLY

PALEOBIOCHEMISTRY AND THE ORIGIN OF MULTICELLULARI

A CENTRAL QUESTION FACING NASA AS IT DESIGNS MISSIONS TO SEARCH FOR LIFE IN THE COSMOS ASKS HOW LIFE MIGHT BE RECOGNIZED IF IT WERE ACTUALLY ENCOUNTE

THIS PROPOSAL MOST DIRECTLY RELATES TO THE PLANETARY SCIENCE/EXOBIOLOGY GOALS THAT SEEK TO UNDERSTAND THE ORIGIN OF LIFE. IT TIES AS WELL TO THE BROADER EXOBIOLOGY PROGRAM IN THE BENNER LAB WHICH EXPLOITS MODELS FOR ORIGINS TO GUIDE THE SEARCH FOR DARWINISM IN THE SOLAR SYSTEM CONSTRUCTS "ALIEN" GENETIC SYSTEMS TO UNDERSTAND THE RANGE OF POSSIBLE BIOSIGNATURES AND MODELS THE EVOLUTION OF TERRAN LIFE WORKING BACK IN TIME TO DEFINE AN "END GOAL" FOR ORIGINS WORK. THUS THIS WORK WILL HAVE IMPACT BEYOND SIMPLY ELABORATING AN IMPORTANT MODEL FOR THE ORIGINS OF LIFE. INDEED SOME OF CHEMISTRY THAT WE DEVELOP COULD BE OCCURRING ON MARS TODAY. THE EXPERIMENTAL WORK IS ORGANIZED AROUND THE "RNA FIRST" MODEL FOR THE ORIGIN OF DARWINISM. IT BUILDS ON THE "DISCONTINUOUS SYNTHESIS MODEL" (DSM) FOR THE PREBIOTIC FORMATION OF RNA. THE DSM IN TURN IS SET IN AN EXPLICIT MODEL FOR EARLY PLANETARY HISTORY INCLUDING A MODEL FOR FORMING THE "LATE VENEER" MODELS FOR THE REDOX POTENTIALS OF THE MANTLE AND ATMOSPHERE AT THE TIME WHEN LIFE EMERGED AND MODELS FOR MINERALS AVAILABLE IN SUCH ENVIRONMENTS. WE THEN IDENTIFY INDIVIDUAL CHALLENGES REMAINING IN THE DSM THAT MUST BE RESOLVED BEFORE IT CAN BE ACCEPTED BY THE COMMUNITY AS ONE POSSIBLE SOLUTION TO THE "ORIGINS" CONUNDRUM. WE THEN PROPOSE EXPERIMENTS WITH ALTERNATIVES TO MANAGE PITFALLS TO RESOLVE THEM. THE WORK BENEFITS FROM SEED MONEY PROVIDED BY THE TEMPLETON FOUNDATION WHICH (TOGETHER WITH NASA SUPPORT) LED TO MULTIPLE DISCOVERIES THAT WILL BE DEVELOPED. WE DISCOVERED: (1) A SPECTRUM OF CARBONATE SULFATE AND SILICATE MINERALS THAT ADSORB AND STABILIZE RNA OFTEN WITH A PERIODIC TABLE TREND; ONE CHIRAL MINERAL (QUARTZ) EVEN DISTINGUISHES HOMOCHIRAL D-RNA AND L-RNA. THIS DISCOVERY WILL BE DEVELOPED BY STUDYING OTHER CHIRAL MINERALS AND MINERAL SURFACES QUANTITATIVELY COMPARING RNA BINDING BY COMPETITION EXPERIMENTS AND MOVING FROM BINARY MINERALS TO TERNARY MINERALS IN REALISTICALLY COMPLEX GEO-ENVIRONMENTS. (2) VOLCANIC SULFUR DIOXIDE PROVIDES A SIMPLE WAY TO GET STABLE RESERVOIRS OF FORMALDEHYDE AND OTHER SIMPLE CARBOHYDRATES; THESE COMPLEMENT BORATE-CARBOHYDRATE MINERALS THAT ALLOW ADVANCED SPECIES TO ACCUMULATE IN USEFUL AMOUNTS. THIS WORK WILL QUANTITATE EQUILIBRIUM CONSTANTS TO ASSESS THE VALUE OF SULFONATES IN MANAGING "TAR" PROBLEMS IN CARBOHYDRATE PREBIOTIC CHEMISTRY ASSESS THEIR COMPATIBILITY WITH OTHER MINERAL SPECIES IN REALISTIC GEO-ENVIRONMENTS AND POSSIBLY RESCUE PROPOSALS (E.G. OF SUTHERLAND) THAT REQUIRE LARGE AMOUNTS OF THESE. (3) WE HYPOTHESIZE THAT MOLYBDENUM (+6) CAN COMPLETE THE MANAGEMENT OF THE "TAR" PROBLEM FOR CARBOHYDRATES; EXPERIMENTS NOW SHOW THAT IT WAS AVAILABLE IN THE HADEAN. WE WILL ASSESS ITS PERFORMANCE IN REALISTICALLY COMPLEX MINERAL ENVIRONMENTS DETERMINE ITS COMPATIBILITY WITH PHOSPHATE BORATE AND OTHER MINERAL ANIONS AND STUDY ITS REDOX INTERACTION WITH FERROUS SULFITE AND OTHER REDUCED SPECIES. (4) MINERAL-BASED PROCESSES USING THE KRISHNAMURTHY POLYPHOSPHATE AMIDE ALLOW FORMATION OF GLYCOSIDIC BONDS OF NUCLEOSIDE PHOSPHATES IN DESERT ENVIRONMENTS; BORATE-MEDIATED REARRANGEMENTS GIVE NUCLEOSIDE 5'-PHOSPHATES. WE WILL ASSESS THREE APPROACHES TO MAKE ACTIVATED NUCLEOSIDE PHOSPHATES AVAILABLE FOR OLIGOMERIC RNA FORMATION INCLUDING ALTERNATIVE OXIDATION STATES OF SULFUR. WE WILL THEN DO "BIG PICTURE" EXPERIMENTS ILLUSTRATING HOW LOEB'S "SILENT DISCHARGE" IN A POST-VENEER ATMOSPHERE CAN PRODUCE SUFFICIENT HCN HNCNH AND OTHER PRODUCTS THAT IF RAINED INTO PHYSICAL ROCK CONTAINING OLIVINE TOURMALINE AND OTHER MINERAL SPECIES GENERATE AN EFFLUENT THAT CAN LEAD TO THE FORMATION OF RNA AFTER DRAINING INTO A DRY SPACE UNDER A CO2 ATMOSPHERE. CONSISTENT WITH A NASA BUDGET THIS WILL START WITH A SYNTHETIC EFFLUENT WITH NUCLEOBASES LIKELY MADE IN GEOTHERMAL REGIONS EXPOSED TO FORMAMIDE CYANAMIDE AND CYANOACETYLENE WITH RNA PRODUCTS STABILIZED BY ABSORPTION ON MINERALS.

SEDIMENTARY MINERALS, ORGANIC CHEMICAL TRANSFORMATIONS, AND THE ORIGIN OF LIFE -OUR UNDERSTANDING OF GEOLOGICAL ENVIRONMENTS ON EARTH 4.35 BILLION YEARS AGO HAS ADVANCED TO THE POINT WHERE GEOLOGY CAN BE COMBINED WITH ORGANIC CHEMISTRY THAT DOES NOT NEED CAREFUL HUMAN CONTROL TO BUILD MODELS FOR HOW THE FIRST GENETIC MOLECULES MIGHT HAVE EMERGED ON EARTH, AND POSSIBLY ON OTHER ROCKY PLANETS LIKE MARS. THIS HANDS OFF CHEMISTRY CAN BE FURTHER SIMPLIFIED IN THE CONTEXT OF ROCKS PRESENT IN THOSE ENVIRONMENTS. THIS COMBINATION NOW ALLOWS US TO ADVANCE SCIENTIFIC KNOWLEDGE RELEVANT TO ONE OF THE OLDEST QUESTIONS POSED BY HUMANKIND: WHERE DID WE COME FROM? FURTHER, THE SIMPLICITY OF THE PREBIOTIC CHEMISTRY THAT THIS PROJECT WILL DEVELOP MAKES IT ACCESSIBLE TO STUDENTS, EVEN THOSE IN HIGH SCHOOL. IN ADDITION TO ADVANCING THE SCIENCE, THIS PROJECT WILL DELIVER KITS TO THOSE STUDENTS THAT WILL LET THEM PARTICIPATE IN THE CURRENT EXCITEMENT IN GEOCHEMISTRY RELEVANT TO THIS BIG QUESTION. THIS GEOLOGY-CHEMISTRY COMBINATION SUPPORTS THE RNA FIRST HYPOTHESIS FOR THE ORIGIN OF LIFE, WHICH HOLDS THAT DARWINIAN EVOLUTION EMERGED BASED ON THE ABIOLOGICAL FORMATION OF RIBONUCLEIC ACID THAT SERVED AS ITS FIRST INFORMATIONAL MOLECULE. HERE, THE GEOLOGICAL ENVIRONMENT COMPRISES INTERMITTENTLY DRY, CONSTRAINED AQUIFERS THAT RECEIVE BY RAIN SMALL CARBOHYDRATES STABILIZED BY VOLCANIC SULFUR DIOXIDE, REDUCED NITROGEN-CONTAINING ORGANIC MOLECULES, AND OTHER SPECIES THAT WERE CREATED BY UV AND ELECTRICAL DISCHARGE IN ATMOSPHERES THAT HAD BEEN REDUCED BY IRON FRAGMENTED FROM THE CORES OF IMPACTING BODIES. THE GEOLOGICAL ENVIRONMENT INCLUDES SURROUNDING ROCKS FROM A REDOX NEUTRAL CRUST, INCLUDING BORATE EVAPORITE MINERALS, AS WELL AS BASALTIC GLASS GENERATED BY THE IMPACTS AND VOLCANISM ON THE YOUNG EARTH. THE BASALTIC GLASS CONTAINS DEHYDRATED PHOSPHATES WHICH CAN SERVE AS A CATALYST TO ASSEMBLE RNA. IN THIS SINGLE ENVIRONMENT, SMALL ORGANICS CAN YIELD OLIGOMERIC RNA 100-200 NUCLEOTIDES LONG BY A PROCESS THAT INITIATES WITH BORATE-CONTROLLED MATURATION OF STABILIZED CARBOHYDRATES, PHOSPHORYLATION BY GLASS-DELIVERED POLYPHOSPHATES, REACTION OF RIBOSE CYCLIC PHOSPHATES WITH NUCLEOBASES TO GIVE NUCLEOTIDES, WITH BORATE-MODERATED PHOSPHORYLATION YIELDING NUCLEOSIDE TRIPHOSPHATES. THIS PROJECT WILL COMPLETE STRUCTURE ANALYSIS OF RNA PRODUCTS FORMED FROM TRIPHOSPHATES BY IMPACT GLASS, INTEGRATE CARBOHYDRATE PROCESSING WITH DOWNSTREAM NUCLEOSIDE SYNTHESIS, EXPLORE THE POSSIBILITY OF THIS ENVIRONMENT PRODUCING HOMOCHIRAL PRODUCTS, AND DEVELOP THE SEDIMENTARY AND IGNEOUS GEOLOGY OF RELEVANT MINERALS FOR STUDENTS TO WORK WITH. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

FIREBUG: A LIVING CELL THAT PROPAGATES PLASMIDS BUILT FROM AN ARTIFICIAL GENETIC SYSTEM

INCREASING AMINO ACID DIVERSITY IN IN VITRO TRANSLATION WITH EXPANDED GENETIC ALPHABETS

HIGH QUALITY PROTEINS WITH MULTIPLE POST TRANSLATIONAL MODIFICATIONS - HIGH QUALITY PROTEINS WITH MULTIPLE POST TRANSLATIONAL MODIFICATIONS FOUNDATION FOR APPLIED MOLECULAR EVOLUTION SHUICHI HOSHIKA ABSTRACT THE PROPOSED TECHNOLOGY WILL MAKE, BY IN VITRO TRANSLATION (IVT), PROTEINS THAT HOLD NON-CANONICAL AMINO ACIDS NORMALLY PUT IN ONLY BY POST-TRANSLATIONAL MODIFICATION (PTM). THE IMMEDIATE DELIVERABLE WILL BE TECH- NOLOGY THAT DELIVERS PROTEINS WITH THREE PTM-AAS (ACETYLLYSINE, PHOSPHSERINE, PHOSPHOTYROSINE) INCORPORATED IN MANY, EXACT POSITIONS IN LONG (300 - 1100 AMINO ACID PROTEINS ARE USED) PROTEINS WITH >95% OCCUPANCY. THE NCI ITSELF MOTIVATED THIS PROPOSAL BY ITS CALLS FOR TOOLS TO MAKE SUCH PROTEINS, WHICH CANCER RESEARCHERS NEED THROUGHOUT CANCER PROTEOMICS. TODAY, SUCH PROTEINS ARE AVAILABLE ONLY VIA ISOLATION FROM LIVING EUKARYOTIC CELLS. THESE ARE RARELY PURE. OUR PURE PTM PROTEINS WILL BE USED TO GET ANTIBODIES, IDENTIFY PTM SIGNATURES OF CANCERS, STANDARDIZE QUANTITATIVE IMMUNOASSAYS AS STANDARDS, DISCOVER INHIBITORS AND DRUGS FOR CANCER-RELATED ENZYMES IN THEIR DRUG-RELEVANT FORMS, AND STUDY PROTEIN-PROTEIN INTERACTIONS. THESE WILL BE OBTAINED RAPIDLY AND INEXPENSIVELY IN THEIR OWN LABS (WITH IN VITRO TRANSLATION KITS) OR VIA SERVICE COMPANIES (AS FOR ANTIBODIES). AS PERFORMANCE MEASURES, THE NCI DEFINED "USEABLE AMOUNTS" TO BE "0.5 TO 1 MG OF PROTEIN" WITH "50- 80% MODIFICATION AT THE SPECIFIED SITE". OUR TECHNOLOGY WILL DO BETTER, GENERATING 1-10 MG OF PROTEIN WITH >95% MODIFICATION FOR THREE DIFFERENT PTM AMINO ACIDS AT MANY SPECIFIED SITES. BEHIND THIS PROJECT IS AN ONGOING REVOLUTION IN THE SYNTHETIC BIOLOGY OF DNA AND RNA (XNA) THAT DELIVERED EXPANDED XNA, ENHANCED IN 2019 BY THE PI. ARTIFICIAL XNA LOOKS LIKE STANDARD XNA. HOWEVER, IT ADDS PAIRS BY SHUFFLING HYDROGEN BONDING GROUPS, ALLOWING EXPANDED XNA TO “WRITE” MORE AA “WORDS” IN A PROTEIN “LEXICON”. CONSISTENT WITH AN IMAT R21 FORMAT, THIS PROJECT WILL PROVE CONCEPTS, DE-RISK PROCEDURES, AND TAKE ENOUGH STEPS TO GUIDE THE NCI AS IT SEEKS TO COMPLETE THIS TRANSFORMATIVE TECHNOLOGY. WE WILL USE ONLY 8 "HACHIMOJI" NUCLEOTIDES TO SYSTEMATICALLY ADD, IN THREE AIMS, THESE PTM-AAS WHILE DEVELOPING THE CONCEPTS TO CONTROL THE INTERACTIONS THAT MUST BE CONTROLLED TO MEET THIS GRAND CHALLENGE: (I) BETWEEN HACHIMOJI CODONS AND ANTICODONS DURING TRANSLATION, (II) BETWEEN SYNTHETASES AND HACHIMOJI ANTICODONS DURING AMINOACYLATION, AND (III) BETWEEN ORTHOGONAL HACHIMOJI CHARGED TRNAS AND PARTS OF THE E. COLI RIBOSOME COMPLEX. EVEN WITH THIS LIMITED SCOPE, THE TECHNOLOGY WILL BE TRANSFORMATIVE BECAUSE OF THE IMPORTANCE OF THESE PTM-AAS. AS AIMS ARE MET, OUR ABILITY TO MEET PERFORMANCE MEASURES WILL BE SHOWN BY MAKING PROHIBITIN 2 (7 EXEMPLARS OF THESE 3 PTM-AAS) USING ENHANCED IVT (EIVT) . AS A LONG TERM DELIVERABLE, SINCE 8-LETTER DNA CAN DELIVER 512 CODONS, ALL PTM AAS CAN BE INCORPORATED INTO PROTEINS USING THIS TECHNOLOGY, A MORE TRANSFORMATIVE OUTCOME. NEXT, AS RESEARCHERS ADVANCE IVT AND MOVE HACHIMOJI DNA INTO LIVING CELLS, EVEN MORE TRANSFORMATIVE OUTCOMES ARE POSSIBLE IN A LONG TERM VISION.

EVOLVED MOLECULES THAT DESTROY CANCER RELEVANT PROTEINS - ABSTRACT CANCER RESEARCH IS OFTEN DRIVEN BY HYPOTHESES THAT POSTULATE THAT A SPECIFIC PROTEIN IS IMPORTANT FOR THE DISEASE, HYPOTHESES OFTEN TESTED BY ADDING AN ANTIBODY TO THAT SYSTEM. THE TECHNOLOGY TO BE DEVELOPED HERE WILL CREATE NEW REAGENTS (AEGISBODIES AND AEGISCLEAVERS) THAT NOT ONLY BIND A TARGET PROTEIN (LIKE AN ANTIBODY), BUT ALSO CLEAVE IT, AT A COST 10 TO 100 FOLD LOWER THAN A SERVICE-PROVIDED ANTIBODY, IN WEEKS RATHER THAN MONTHS, AND ACROSS A RANGE OF AFFINITIES THAT SUPPORT QUANTITATIVE RESEARCH. THESE WILL BE OBTAINED BY APPLYING LABORATORY IN VITRO EVOLUTION (LIVE) TO LIBRARIES BUILT FROM AN ARTIFICIALLY EXPANDED GENETIC INFORMATION SYSTEM (AEGIS), AND DECORATED WITH PRECISELY SELECTED CHEMICAL GROUPS. AEGIS IS A BIOPOLYMER LIKE DNA, BUT WITH UP TO 12 BUILDING BLOCKS, ENHANCED FOLDING, GREATER STABILITY, AND FUNCTIONAL GROUPS THAT ASSIST IN THE CLEAVAGE REACTION, WHICH GIVES PRODUCTS WHERE AN AEGISCLEAVER IS COVALENTLY ATTACHED TO A FRAGMENT OF THE TARGET PEPTIDE. AEGIS-LIVE HAS HIGH LEVEL OF TECHNICAL READINESS BECAUSE OF PRELIMINARY STUDIES THAT CREATED (A) PLATFORMS TO MANUFACTURE AEGIS DNA, (B) A MOLECULAR BIOLOGY TO SUPPORT AEGIS-LIVE, AND (C) SEQUENCING, FOLD PREDICTION, AND OTHER TOOLS TO ANALYZE THE PRODUCTS OF AEGIS-LIVE. THE TEAM HAS PROVEN THE CANCER-RELEVANCE OF AEGIS-LIVE, CREATING AEGISBODIES THAT BIND SPECIFICALLY AND SELECTIVELY TO BREAST AND LIVER CANCER CELLS, TO CANCER-RELEVANT PROTEINS HETEROLOGOUSLY EXPRESSED ON MAMMALIAN CELL SURFACES, AND TO ISOLATED PROTEINS FROM ANTHRAX. THE FIRST CATALYTIC AEGISZYMES ARE IN HAND. TO FURTHER THIS TECHNOLOGY DEVELOPMENT, WE WILL: AIM 1. APPLY AEGIS-LIVE TO CREATE AEGISBODIES THAT BIND PROGRAMMED DEATH-LIGAND 1 (PD-L1), A RECOGNIZED TARGET FOR CANCER THERAPY. WE WILL ALSO TARGET EXTRACELLULAR PEPTIDE LOOPS IN THE PD-L1 FOLD, MADE FROM L- AND D- AMINO ACIDS. THE SECOND ARE MIRROR IMAGES OF THE NATURAL AMINO ACIDS, AND ALLOW PRODUCTION OF MIRROR IMAGE L- AEGISBODIES THAT ARE STABLE AGAINST NUCLEASE DIGESTION. WE WILL METRIC THESE FOR BINDING AFFINITIES AND STABILITY, AND VALIDATE THEIR EFFICACIES COMPARED TO CURRENT TECHNOLOGIES. AIM 2. APPLY AEGIS-LIVE TO CREATE L- AND D-AEGISCLEAVERS THAT CLEAVE PEPTIDES FROM PD-L1, AND THE ANALOGOUS SEGMENTS WHEN EMBEDDED IN THE COMPLETE PROTEIN. WE WILL METRIC AND BENCHMARK THESE FOR AFFINITY, CLEAVAGE RATES, AND STABILITY AGAINST NUCLEASES. THE NEW REAGENTS WILL BE TRANSFORMATIVE SINCE THEY DO THINGS THAT ANTIBODIES CANNOT: COVALENTLY TAG AND DESTROY TARGET PROTEINS. FURTHER, THE REAGENTS ARE EXPECTED TO BE STABLE IN BIOLOGICAL FLUIDS, INCLUDING CELL CULTURE, BLOOD, AND LIVING ANIMALS. LONGER TERM, ULTRA-STABLE AEGISCLEAVERS MAY EVEN COME TO BE DIAGNOSTIC AND THERAPEUTIC TOOLS.

DIVERSE EVOLUTIONARY POWER OF NUCLEIC ACID LIBRARIES CARRYING DIFFERENT INFORMATION CONTENT

TECHNOLOGY TO CREATE SPIEGEL ERABODIES ON DEMAND: BIOSTABLE UNIVERSAL ANTIBODY REPLACEMENTS - TECHNOLOGY TO CREATE SPIEGEL ERABODIES ON DEMAND: BIOSTABLE UNIVERSAL ANTIBODY REPLACEMENTS FOUNDATION FOR APPLIED MOLECULAR EVOLUTION ELISA BIONDI ABSTRACT RESEARCHERS IN BIOMEDICAL, DIAGNOSTIC, AND CLINICAL AREAS WANT TO CREATE (OR BUY), ON DEMAND, REAGENTS THAT BIND TO PROTEINS AND OTHER TARGETS THAT MAY BE INVOLVED IN A BIOLOGICAL PROCESS THAT THEY ARE STUDYING. ANTIBOD- IES HAVE LONG SERVED THIS ROLE. HOWEVER, AS BIOLOGICS, ANTIBODIES ARE AT THE CENTER OF AN "IRREPRODUCIBILITY CRISIS" IN BIOMEDICAL RESEARCH, AND, EVEN WHEN SUITABLE, TAKE MONTHS AND THOUSANDS OF DOLLARS TO MAKE. THIS HAS DRIVEN EFFORTS TO CREATE ANTIBODY REPLACEMENTS, BOTH PROTEIN (E.G. DARPINS) AND RNA (E.G. APTAMERS). THE FIRST ARE DIFFICULT TO MANIPULATE, WHILE THE SECOND HAVE LOW STABILITY AND DISAPPOINTING AFFINITY. WE HYPOTHESIZE THAT AN UNNATURAL PLATFORM WITH AN "EXPANDED RNA ALPHABET" (ERA) AND EXTRA FUNCTIONAL GROUPS WITH EXTRA BINDING POTENTIAL WILL MEET THIS LONG-STANDING UNMET NEED. WHILE EXPANDED DNA ALPHABETS ARE NOW ADVANCED, A FIRST INNOVATION IS THAT ERAS HAVE NOT YET BEEN THE TARGET OF ANY PRELIMINARY DATA. WE HYPOTHESIZE THAT NANOMOLAR BINDING WILL BE ROUTINELY ACHIEVED BECAUSE ERABODIES WILL HAVE ACCESS TO (A) HIGHER INFORMATION DENSITY THAT WILL LEAD TO (B) BETTER DEFINED FOLDS, BOTH BY USING AN RNA SCAFFOLD AND BY HAVING FUNCTIONALITY THAT SUPPORTS FOLDING, (C) GREATER STRUCTURAL DIVERSITY THAT GIVES ERABODIES MORE MODES FOR TIGHT BINDING, AND (D) MORE FOLDING MOTIFS THAT ALLOW ERABODIES TO HAVE MORE COMPACT STRUCTURES. THEY ARE ALSO HYPOTHESIZED TO HAVE ALL OF THE ADVANTAGES OF CLASSICAL APTAMERS, INCLUDING VALUE AS THE STARTING POINTS FOR SUBSEQUENT ROUNDS OF EVOLUTION, MODIFIABILITY USING SIGNALING ENTITIES, LOW COST, FAST TURNAROUND, AND DIRECT CHEMICAL SYNTHESIS. THIS R21 PROJECT WILL PROVE THE VALUE OF THIS NEW TECHNOLOGY PLATFORM, WHICH WILL ALLOW RESEARCHERS TO ORDER OR DIRECTLY CREATE IN WEEKS, BINDERS FOR TARGETS THAT THEY THEMSELVES SELECT. WE HYPOTHESIZE A FURTHER INNOVATION BY MERGING YET UNEXPLORED ERA TECHNOLOGY WITH THE CLASSICAL CONCEPT OF MIRROR SYMMETRY. TO CREATE ERABODIES THAT ARE STABLE IN BIOLOGICAL SYSTEMS, WE WILL MAKE THESE IN MIRROR IMAGE ("SPIEGEL") FORM. AIM 1. A SINGLE R21 DEMONSTRATION PROJECT WILL SHOW THAT ERABODIES CAN BE MADE WITH BUILDING BLOCKS CREATED BY PALLADIUM CHEMISTRY. ALTHOUGH THIS TECHNOLOGY IS AGNOSTIC WITH RESPECT TO APPLICATIONS (IT CAN CREATE BINDERS FOR ANY TARGET), THIS DEMONSTRATION WILL HAVE IMPACT BY TARGETING A PROTEIN WITH OUTSIZED MEDICAL SIGNIFICANCE, PD-L1 (PROGRAMMED DEATH LIGAND 1). WE WILL TEST THE HYPOTHESIS THAT L-ERA REAGENTS, MUCH LIKE STANDARD L-RNA, ARE STABLE AGAINST RNASES IN LIVING SYSTEMS. THIS WORKFLOW STEP WILL CONSUME 18 MONTHS. WE WILL EXPAND METHODS TO SEQUENCE ERA AND BENCHMARK THE FIDELITY OF ENZYMATIC SYNTHESIS. IN THE WORKFLOW, THIS WILL BE COMPLETED IN THE FIRST 6 MONTHS, AS PATTERNS OF TRANSLITERATION BY VARIOUS REVERSE TRANSCRIPTASES WILL BE USED TO DEFINE A SEQUENCING PROCEDURE APPLICABLE TO VARIOUS 6- AND 8-LETTER ERA SYSTEMS. THE TARGET METRICS ARE BINDING AFFINITY (NANOMOLAR), BINDING SPECIFICITY (100:1 DISCRIMINATION), AND IN VITRO SERA STABILITY (<0.1% RNASE DEGRADATION OVER 2 HOURS).

POINT-OF-SAMPLING DETECTION OF HUMAN PAPILLOMAVIRUS (HPV)

NUCLEIC ACID INNOVATIONS TO MANAGE PATHOGEN SEQUENCE DIVERGENCE

POINT-OF-SAMPLING DETECTION OF LYME AND OTHER TICK-BORNE DISEASES

IN SILICO AND IN VITRO INVESTIGATION OF NON-CONSERVED INTERACTION CHARACTERISTICS

ROL:EAGER: DESYN-C3. BOTTOM-UP SYNTHETIC CELLS CAPABLE OF DARWINISM, THE ARCHETYPAL TRAIT OF LIFE

OPUS: GEOLOGICAL, ENVIRONMENTAL, AND CHEMICAL BIOLOGY

ADD ON TO LARGE SCALE WATER MINING OPERATIONS ON MARS TO SCREEN FOR INTRODUCED AND ALIEN LIFE