THE PROPOSED RESEARCH IS PART OF A NASA-SPONSORED EFFORT TO UNDERSTAND THE ORIGIN OF LIFE ON EARTH WHICH WILL GUIDE THE SEARCH FOR LIFE ELSEWHERE IN THE UNIVERSE. THE RESEARCH PERTAINS TO A FORM OF RNA-BASED LIFE THAT LIKELY EXISTED DURING EARTH S EARLY HISTORY AS A PREDECESSOR TO CURRENT DNA-RNA-PROTEIN-BASED LIFE. BUILDING ON PROGRESS MADE OVER THE PRIOR GRANT PERIOD THE CENTRAL AIM OF THE PROPOSED RESEARCH IS TO CONSTRUCT RNA-BASED LIFE IN THE LABORATORY AS REPRESENTED BY POPULATIONS OF RNA ENZYMES THAT CATALYZE THEIR OWN REPLICATION AND UNDERGO DARWINIAN EVOLUTION IN A SELF-SUSTAINED AND OPEN-ENDED MANNER. MOST OF THESE EFFORTS WILL FOCUS ON AN RNA POLYMERASE RIBOZYME THAT IS NOW ABLE TO SYNTHESIZE FUNCTIONAL RNAS AND TO CATALYZE THE EXPONENTIAL AMPLIFICATION OF SHORT RNAS. THE ENZYME IS NOT YET ABLE TO SYNTHESIZE ITSELF BUT THAT GOAL IS LIKELY TO BE ACHIEVABLE WITH ADDITIONAL OPTIMIZATION PRIMARILY BY EMPLOYING IN VITRO EVOLUTION METHODS TO ENHANCE THE RATE FIDELITY AND SEQUENCE GENERALITY OF THE POLYMERASE. ALTHOUGH THE AIM IS TO SYNTHESIZE THE POLYMERASE IN ITS ENTIRETY A DIVIDE-AND-CONQUER APPROACH ALSO WILL BE PURSUED WHEREBY THE ENZYME WILL BE DIVIDED INTO FRAGMENTS THAT CAN ASSEMBLE NON-COVALENTLY TO FORM A CATALYTIC COMPLEX WITH EACH OF THE FRAGMENTS (AND THEIR COMPLEMENTS) SYNTHESIZED BY THE ASSEMBLED COMPLEX. THIS APPROACH MAY REFLECT WHAT TRANSPIRED ON THE PRIMITIVE EARTH BUT ALSO EXPLORES A GENERAL PRINCIPLE FOR HOW DARWINIAN SYSTEMS MIGHT ARISE. THE DIVIDE-AND-CONQUER APPROACH HAS ALREADY BEEN USED FOR THE POLYMERASE RIBOZYME TO SYNTHESIZE ITS EVOLUTIONARY ANCESTOR AN RNA LIGASE RIBOZYME WHICH CAN BE DIVIDED INTO FRAGMENTS THAT ARE SYNTHESIZED SEPARATELY AND CAN ASSEMBLE NON-COVALENTLY TO FORM A FUNCTIONAL CATALYST. THIS APPROACH WILL BE EXTENDED TO THE SYNTHESIS OF LARGER AND MORE COMPLEX RNA ENZYMES INCLUDING THE POLYMERASE ITSELF. A SURPRISING DISCOVERY DURING THE PRIOR GRANT PERIOD WAS THAT HIGHLY OPTIMIZED FORMS OF THE RNA POLYMERASE RIBOZYME ALSO CAN FUNCTION AS A REVERSE TRANSCRIPTASE COPYING RNA TEMPLATES TO COMPLEMENTARY DNA PRODUCTS. THIS ACTIVITY WOULD HAVE BEEN CRUCIAL FOR THE TRANSITION FROM RNA TO DNA GENOMES DURING THE EARLY HISTORY OF LIFE. MORE RECENTLY IT WAS SHOWN THAT THE POLYMERASE CAN COPY DNA TEMPLATES TO RNA PRODUCTS AND AT LOWER EFFICIENCY COPY DNA TEMPLATES TO DNA PRODUCTS. FURTHERMORE RNA TEMPLATES CAN BE COPIED TO YIELD VARIOUS POLYNUCLEOTIDE ANALOGS MOST NOTABLY THREOSE NUCLEIC ACID (TNA) WHICH HAS BEEN SUGGESTED AS A POSSIBLE PREDECESSOR TO RNA. THESE ACTIVITIES WILL BE OPTIMIZED USING IN VITRO EVOLUTION TO GENERATE A FAMILY OF POLYMERASE RIBOZYMES THAT CAN BOTH AMPLIFY AND TRANSCRIBE MACROMOLECULAR INFORMATION IN VARIOUS FORMS. ALTHOUGH LIFE ON EARTH CAME TO ADOPT RNA AND ULTIMATELY DNA AS THE GENETIC MATERIAL A BROADER VIEW IS REQUIRED WHEN CONSIDERING THE POSSIBILITY OF LIFE ELSEWHERE. ONCE A SYSTEM IS IN HAND FOR THE SELF-SUSTAINED EVOLUTION OF RNA IT WILL BE POSSIBLE TO CONDUCT OPEN-ENDED RNA EVOLUTION EXPERIMENTS BOTH AS A WORKING MODEL OF RNA-BASED LIFE AND AS A MEANS TO EXPLORE THE DIVERSIFICATION OF RNA FUNCTION. THE PROMISE OF SUCH EXPERIMENTS WAS EXAMINED PREVIOUSLY USING A SELF-REPLICATING LIGASE RIBOZYME WHICH COULD BE MADE TO ADAPT TO DIFFERENT SELECTION CONSTRAINTS. HOWEVER THAT SYSTEM HAS A VERY LIMITED CAPACITY TO EVOLVE AND IS NOT CAPABLE OF INVENTING NOVEL FUNCTION. IN CONTRAST THE POLYMERASE ENZYME SUPPORTS MUCH HIGHER INFORMATION CONTENT AMONG THE RNAS BEING REPLICATED. IT IS ALREADY CAPABLE OF SYNTHESIZING COMPLEX FUNCTIONAL RNAS WITH GOOD FIDELITY AND ITS CAPABILITIES WILL BE ENHANCED THROUGH FURTHER OPTIMIZATION. ULTIMATELY WHEN THE ENZYME HAS THE ABILITY TO GENERATE COPIES OF ITSELF THE SYSTEM WILL BECOME SELF-OPTIMIZING BOTH FOR WHAT IT EVOLVES AND FOR ITS CAPACITY TO EVOLVE. IT HAS THE POTENTIAL TO CAPTURE THE DEFINING FEATURE OF LIFE AND TO PROVIDE THE FIRST EXAMPLE OF A LIVING SYSTEM OUTSIDE OF TERRESTRIAL BIOLOGY.