INTRODUCTION: THE OVERALL OBJECTIVE OF THE PROPOSED RESEARCH IS TO INVESTIGATE HOW AN EARLY LIFE FORM COULD HAVE EMERGED ON EARLY EARTH OR OTHER PLANETS. THE FOCUS IS ON THE RNA WORLD HYPOTHESIS WHICH STATES THAT TODAY'S DNA/RNA/PROTEIN LIFE FORMS WERE PRECEDED BY ORGANISMS USING RNA AS GENOME AND AS ONLY GENOME-ENCODED CATALYST. IT IS CURRENTLY UNCLEAR HOW SUCH AN RNA WORLD ORGANISM COULD HAVE FUNCTIONED AND HOW IT COULD HAVE EMERGED FROM A PREBIOTIC ENVIRONMENT. KEY OBJECTIVES OF THE PROPOSED RESEARCH: THE PROPOSED RESEARCH WILL STUDY WHAT CONDITIONS COULD MAKE CATALYTIC RNAS (RIBOZYMES) LIKELY TO EMERGE FROM A PREBIOTIC ENVIRONMENT. LONG RNA POLYMERS ARE UNLIKELY TO HAVE EXISTED IN A PREBIOTIC ENVIRONMENT AND SHORTER RNA OLIGOMERS WERE MUCH MORE FREQUENT. TWO STRATEGIES WILL BE STUDIED THAT COULD HAVE LED TO RIBOZYMES FROM SHORT OLIGOMERS: THE ASSEMBLY OF LARGER RIBOZYMES FROM SHORT RNA OLIGOMERS AND THE SIZE REDUCTION OF RIBOZYMES BY FINE-TUNING RNA DUPLEX STABILITY. METHODS / TECHNIQUES USED IN THE PROPOSED RESEARCH: THE PROPOSED RESEARCH WILL USE AN IN VITRO SELECTION SYSTEM WE PREVIOUSLY ESTABLISHED IN OUR LAB TO IDENTIFY RIBOZYMES UNDER DIFFERENT CONDITIONS AND TO IDENTIFY RIBOZYMES COMPOSED OF MULTIPLE SHORT STRANDS. HIGH THROUGHPUT SEQUENCING (HTS) ANALYSIS WILL BE USED FOR THE ANALYSIS OF THE OUTCOME OF THE IN VITRO SELECTIONS. RNA BIOCHEMISTRY AND MOLECULAR BIOLOGY TECHNIQUES WILL BE USED THROUGHOUT THE SELECTIONS AND THE ANALYSIS OF RESULTING RIBOZYMES. THOSE METHODS INCLUDE PCR (POLYMERASE CHAIN REACTION) IN VITRO TRANSCRIPTION PURIFICATION BY DENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) CLONING AND SEQUENCING RADIOLABELING OF RNA MOLECULES AND QUANTITATIVE ANALYSIS OF THE RADIOLABELED REACTION PRODUCTS. PERCEIVED SIGNIFICANCE OF THE PROPOSED WORK TO THE OBJECTIVES OF THE RESEARCH SOLICITATION AND TO NASA INTERESTS AND PROGRAMS IN GENERAL: THE PROPOSED RESEARCH IS SIGNIFICANT FOR NASA'S RESEARCH EMPHASIS 'PREBIOTIC EVOLUTION' IN AT LEAST THREE WAYS. FIRST THE RESULTS OF ALL THREE AIMS COULD IDENTIFY NEW TYPES OF CATALYTIC RNAS THAT WOULD HAVE BEEN MORE LIKELY TO ARISE IN A PREBIOTIC ENVIRONMENT. THIS IS IMPORTANT FOR NASA'S OBJECTIVE TO DETERMINE "WHAT CHEMICAL SYSTEMS COULD HAVE SERVED AS PRECURSORS OF METABOLIC AND REPLICATING SYTEMS ON EARTH AND ELSEWHERE". SECOND THE IN VITRO SELECTIONS AT LOWER TEMPERATURE WILL EXPLORE WHETHER LOWER TEMPERATURES COULD FAVOR THE EMERGENCE OF RNA CATALYSTS. THIS WILL REPORT WHETHER A 'COLD RNA WORLD' WOULD HAVE HELPED ITS EMERGENCE FROM APREBIOTIC SCENARIO IN LINE WITH NASA'S GOAL TO EXPLORE THE "PHYSICAL AND CHEMICAL CONDITIONS WITHIN WHICH LIVING SYSTEMS MAY HAVE ARISEN." THIRD THE LONG-TERM GOAL OF THE PROPOSED RESEARCH IS TO GENERATE A SELF-REPLICATING AND EVOLVING SYSTEM OF NUCLEIC ACIDS IN THE LAB. THEREFORE IN THE LONG TERM THE RESULTS OF THE PROPOSED RESEARCH ADDRESS THE QUESTION "WHAT CHEMICAL SYSTEMS COULD HAVE SERVED AS PRECURSORS OF METABOLIC AND REPLICATING SYTEMS ON EARTH AND ELSEWHERE".