VISION: AN OUTSTANDING QUESTION IN THE ORIGIN OF LIFE IS HOW PROTOCELLS COMPOSED OF A MEMBRANE RNA AND PROTEIN AROSE ON THE EARLY EARTH. OUR PROPOSED EXPERIMENTS TEST KEY ELEMENTS OF A COMPELLING SCENARIO FOR THE EMERGENCE OF PROTOCELLS IN WHICH: - THE BUILDING BLOCKS OF RNA AND PROTEIN WERE SELECTED AND CONCENTRATED VIA BINDING TO FATTY ACID AGGREGATES WHICH SELF-ASSEMBLE IN WATER. - THOSE BOUND BUILDING BLOCKS STABILIZED FATTY ACID MEMBRANES AGAINST SALT-INDUCED FLOCCULATION AND THE MEMBRANE SURFACES IN TURN ORIENTED THE BUILDING BLOCKS TO FACILITATE THEIR POLYMERIZATION. - MEMBRANES THAT WERE MORE STABLE BOUND MORE BUILDING BLOCKS LEADING TO AN AUTO-AMPLIFYING SYSTEM. - POLYMERS OF THE BUILDING BLOCKS STABILIZED MEMBRANES MORE EFFECTIVELY THAN INDIVIDUAL COMPONENTS DID EXPLAINING THEIR ACCUMULATION PRIOR TO THE EVOLUTION OF THEIR COMPLEX FUNCTIONS IN METABOLISM AND INFORMATION TRANSFER. OUR TEAM HAS A HISTORY OF PRODUCTIVE COLLABORATION AND HAS ESTABLISHED FEASIBILITY OF THE PROPOSED PROJECT. SPECIFICALLY WE PREVIOUSLY SHOWED THAT THE BUILDING BLOCKS OF RNA NUCLEOBASES AND SUGARS DO INDEED BIND TO MEMBRANES AND INCREASE MEMBRANE STABILITY (PNAS 2013). CENTRAL OBJECTIVES: EACH OF OUR OBJECTIVES RELATES TO A THEME OF BROAD INTEREST IN PREBIOTIC EVOLUTION. (1) TEST WHETHER THE ASSOCIATION OF PREBIOTIC FATTY ACIDS WITH NUCLEOBASES AND RIBOSE FACILITATES THE CRITICAL PREBIOTIC REACTIONS OF RIBONUCLEOTIDE SYNTHESIS AND POLYMERIZATION. (HOW DID RNA ARISE?) (2) TEST WHETHER AMINO ACIDS AND PEPTIDES INTERACT WITH FATTY ACID MEMBRANES AND IF SO WHETHER THIS INTERACTION FACILITATES PEPTIDE BOND FORMATION. (HOW DID PROTEINS ARISE?) (3) TEST WHETHER BASES SUGARS AMINO ACIDS NUCLEOTIDES AND COMBINATIONS THEREOF LOWER THE CRITICAL VESICLE CONCENTRATION (CVC) OF FATTY ACIDS OR INCREASE VESICLE GROWTH RATES. (HOW DID STABLE MEMBRANES ARISE?) (4) TEST WHETHER DINUCLEOTIDES AND/OR DIPEPTIDES LOWER THE CVC OF A FATTY ACID OR INCREASE THE GROWTH RATE OF VESICLES MORE THAN UNLINKED NUCLEOTIDES OR AMINO ACIDS DO. (HOW WERE OLIGONUCLEOTIDES AND PEPTIDES SELECTED BEFORE THEY GREW LONG ENOUGH TO BE FUNCTIONAL CATALYSTS AND CARRIERS OF INFORMATION?) METHODS: WE WILL ACCOMPLISH OUR OBJECTIVES USING ONLY PREBIOTICALLY PLAUSIBLE MOLECULES (E.G. DECANOIC ACID) TO ENSURE THAT OUR RESULTS ARE DIRECTLY APPLICABLE TO THE ORIGINS OF LIFE. WE WILL EMPLOY STANDARD IN VITRO ASSAYS LISTED BELOW AS WELL AS NOVEL ASSAYS DEVELOPED BY OUR GROUP. - FOR OBJECTIVE 1: WE WILL TEST FOR NUCLEOSIDE AND OLIGONUCLEOTIDE FORMATION IN THE PRESENCE OF (A) POTENTIAL PREBIOTIC CATALYSTS (E.G. PHOSPHATE AMINO ACIDS AND DIPEPTIDES) (B) UV RADIATION AND (C) CYCLIC DEHYDRATION AT VARIOUS TEMPERATURES. WE WILL TEST FOR SUCCESSFUL SYNTHESIS USING TLC HPLC MASS SPECTROMETRY AND AGAROSE GEL ELECTROPHORESIS. - FOR OBJECTIVE 2: WE WILL USE THE FILTRATION AND FLOCCULATION ASSAYS WE PREVIOUSLY DEVELOPED (PNAS 2013) TO EVALUATE BINDING OF AMINO ACIDS AND PEPTIDES TO FATTY ACID VESICLES. WE WILL INCUBATE AMINO ACIDS WITH VESICLES UNDER THE CONDITIONS AS DESCRIBED FOR OBJECTIVE 1 AND THEN SCREEN FOR PEPTIDES BY TLC AND MASS SPECTROMETRY. - FOR OBJECTIVES 3 AND 4: WE WILL MEASURE CVC USING TURBIDITY AND PINACYANOL CHLORIDE ABSORBANCE ASSAYS AND WE WILL MEASURE GROWTH RATES USING DYNAMIC LIGHT SCATTERING. SIGNIFICANCE: THIS PROJECT ADDRESSES A CENTRAL THEME OF THE 2016 NASA EXOBIOLOGY PROGRAM IN SECTION C.5 ON PREBIOTIC EVOLUTION. WE WILL INVESTIGATE THE SELF-ASSEMBLY OF MEMBRANES AND THE BUILDING BLOCKS OF RNA AND PROTEIN AS A MOLECULAR [PROCESS] THAT SET THE PHYSICAL AND CHEMICAL CONDITIONS WITHIN WHICH LIVING SYSTEMS MAY HAVE ARISEN . ESTABLISHING CONDITIONS UNDER WHICH THE ASSEMBLED COMPONENTS FORM POLYMERS WILL REVEAL ENVIRONMENTAL REQUIREMENTS FOR THE EMERGENCE OF PROTOCELLS. THE PROJECT WILL DIRECTLY DEMONSTRATE A CHEMICAL SYSTEM THAT COULD HAVE SERVED AS [A] PRECURSOR OF METABOLIC AND REPLICATING SYSTEMS ON EARTH.