PRE-CELLULAR COMPARTMENTALIZATION FOR RNA SYNTHESIS, STRUCTURE, AND FUNCTION

A NUMBER OF THEORIES HAVE BEEN ADVANCED FOR THE ORIGIN OF LIFE MANY OF WHICH POSIT THAT RNA OR RNA-LIKE MOLECULES WERE BOTH INFORMATION-CARRYING AND CATALYTIC ENTITIES. ULTIMATELY COMPARTMENTALIZATION OF THESE FUNCTIONAL MOLECULES WAS REQUIRED TO FORM THE EARLIEST REPLICATING CELLS. THIS PROPOSAL SEEKS TO UNDERSTAND GENERAL PHYSICAL AND CHEMICAL PRINCIPLES THAT LED TO THE FIRST CELLS AND INVESTIGATES PHYSICAL MEANS THAT MAY HAVE FACILITATED THE EMERGENCE OF LIFE THROUGH THE SYNTHESIS OF RNA POLYMERS AND THEIR LOCALIZATION AND CATALYSIS. THE PROPOSED WORK IS WITHIN THE SCOPE OF THE EXOBIOLOGY PROGRAM ELEMENT (C.5 OF ROSES 2016) AND SPECIFICALLY THE PREBIOTIC EVOLUTION RESEARCH EMPHASIS. IT ADDRESSES TOPIC 2 SYNTHESIS AND FUNCTION OF MACROMOLECULES IN THE ORIGIN OF LIFE FROM THE 2015 NASA ASTROBIOLOGY STRATEGY. IT SPEAKS TO KEY QUESTIONS REGARDING EARLY POLYMERS AND BIG PICTURE QUESTIONS ABOUT COUPLING MONOMERIC BUILDING BLOCKS AND CATALYZING REACTIONS IMPORTANT TO EARLY CHEMICAL EVOLUTION INCLUDING SELF-REPLICATION. IT ALSO ADDRESSES SEVERAL KEY RESEARCH QUESTIONS INCLUDING: WHAT IS THE ROLE OF ENVIRONMENT? HOW DID PHYSICOCHEMICAL EFFECTS DEVELOP OVER TIME? WHAT ARE THE ADVANCED STEPS OF MACROMOLECULAR FUNCTION? OUR TEAM BRINGS FRESH INSIGHT BY COMBINING EXPERTISE IN COMPARTMENTALIZATION (KEATING) AND RIBOZYME CHEMISTRY (BEVILACQUA). IN OUR CURRENT FUNDING WE HAVE GENERATED A SERIES OF DIVERSE NON-MEMBRANOUS COMPARTMENTS (NMCS) BY LIQUID-LIQUID PHASE COEXISTENCE IN AQUEOUS POLYMER SOLUTIONS AND USED THESE TO COMPARTMENTALIZE NUCLEOTIDES AND RNA COMPACT RNA STRUCTURE AND ENHANCE RNA POLYMERIZATION AND CATALYSIS. WE DISCOVERED THAT CERTAIN NMCS HAVE REMARKABLE PROPERTIES THAT SHOULD FAVOR THE SYNTHESIS AND FUNCTION OF RNA; THESE OBSERVATIONS UNDERPIN THE WORK PROPOSED HERE. RESEARCH OBJECTIVES: (1) UNDERSTAND AND CONTROL HOW NMC COMPOSITION DICTATES RNA ACCUMULATION DYNAMICS AND STRUCTURE. (2) NMC-FACILITATED RNA POLYMERIZATION AND FUNCTION. (3) NMC-FACILITATED EMERGENCE AND EVOLUTION OF NEW FUNCTIONAL RNAS. APPROACH: OUR NMCS ARE LIQUID DROPLETS FORMED BY PHASE SEPARATION IN AQUEOUS SOLUTIONS OF ONE OR MORE MACROMOLECULAR COMPONENTS. THEY OFFER STRONG SOLUTE ACCUMULATION AND REACTION ENVIRONMENTS DISTINCT FROM THE DILUTE AQUEOUS SURROUNDINGS AND ARE FEASIBLE ON EARLY EARTH BECAUSE THEY OCCUR AT LOW CONCENTRATIONS FOR MANY DIFFERENT POLYMER CHEMISTRIES. NMC OCCURRENCE COMPOSITION AND PHYSICAL PROPERTIES WILL BE EVALUATED AS A FUNCTION OF THEIR MAKEUP AND SOLUTION ENVIRONMENT (SALT PH TEMPERATURE). RNA MOBILITY WITHIN THE COMPARTMENTS AND RNA EXCHANGE WITH THE EXTERNAL SOLUTION WILL BE DETERMINED BY DIFFUSION MEASUREMENTS. PARTITIONING WILL BE ASSAYED BY SCINTILLATION COUNTING USING RADIOLABELED RNAS AND FOR INDIVIDUAL DROPLET-LEVEL STUDIES BY FLUORESCENCE USING DYE-LABELED RNAS. RNA SYNTHESIS FUNCTION AND STRUCTURE WILL BE TESTED IN THE NMCS AS WELL. SYNTHESIS WILL BE ASSAYED BY GEL ELECTROPHORESIS OF LABELED RNAS. RIBOZYME FUNCTION WILL BE TESTED BY STANDARD KINETIC ASSAYS WITH RADIOISOTOPE OR FRET LABELS. STRUCTURE OF FUNCTIONAL RNAS WILL BE ASSESSED AT THE ATOMIC LEVEL BY IN-LINE PROBING OR SHAPE; AT THE BASE PAIRING LEVEL BY FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) WHICH CAN BE DONE IN BULK OR AT THE INDIVIDUAL DROPLET LEVEL; AND AT THE GLOBAL LEVEL BY HYDROXYL RADICAL FOOTPRINTING SAXS AND KINETIC ASSAYS. SIGNIFICANCE: THE WORK PROPOSED HERE WILL ESTABLISH PRINCIPLES THAT MAY HAVE LED TO THE FIRST CELLS AND WILL ELUCIDATE HOW THESE PROTOCELL COMPARTMENTS WOULD HAVE FACILITATED THE SYNTHESIS STRUCTURE AND FUNCTION OF RNA.