GOALS THE RNA WORLD HYPOTHESIS PREDICTS THAT FUNCTIONAL RNA MOLECULES PLAYED A CENTRAL ROLE IN THE ORIGINS AND EARLY EVOLUTION OF LIFE. TO CRITICALLY EVALUATE THIS SCENARIO WE NEED TO UNDERSTAND THE CAPACITY OF POPULATIONS OF RNA MOLECULES TO EVOLVE SPECIFIC ADAPTATIONS. CURRENTLY THE MOST THOROUGH APPROACH IS TO EVALUATE VAST LIBRARIES OF SEQUENCES USING NEXT GENERATION SEQUENCING. BY ASSIGNING A RELATIVE ACTIVITY (FITNESS) TO EACH SEQUENCE IT IS POSSIBLE TO CONSTRUCT EMPIRICAL FITNESS LANDSCAPES. THESE LANDSCAPES CAN BE IMAGINED IN THREE DIMENSIONS WITH THE X-Y-PLANE REPRESENTING NUCLEOTIDE CHANGES (GENOTYPE SPACE) AND THE Z-AXIS REPRESENTING RNA FITNESS. HOWEVER FOR A GIVEN RNA SEQUENCE THIS FITNESS VALUE WILL CHANGE IF THE CONDITIONS OF THE EXPERIMENT CHANGE (ENVIRONMENTAL CHANGE). IMPORTANTLY ENVIRONMENTAL CHANGES MAY PROMOTE ADAPTATION FOR EXAMPLE IF LOW FITNESS VALLEYS BECOME CROSSABLE WHEN THE LANDSCAPE CHANGES. IN THE ABSENCE OF MODERN CELLULAR LIFE CHANGES IN THE PHYSICOCHEMICAL ENVIRONMENT WOULD HAVE BEEN CONSTANT AND UNBUFFERED. DESPITE THE IMPORTANCE OF ENVIRONMENTAL FLUCTUATIONS FOR RNA FITNESS LANDSCAPES THE RELATIONSHIP BETWEEN FITNESS LANDSCAPES AND CHANGES IN THE ENVIRONMENT REMAINS UNDERSTUDIED. HERE WE PROPOSE TO EVALUATE CHANGES IN RNA FITNESS LANDSCAPES ACROSS CHEMICAL GRADIENTS. WE WILL UTILIZE NEXT GENERATION SEQUENCING AS A HIGH-THROUGHPUT BIOCHEMICAL ASSAY. WE WILL SPREAD SOME OF THE THROUGHPUT OVER MULTIPLE POINTS ON AN ENVIRONMENTAL GRADIENT. WE WILL USE NON-LINEAR CURVE FITTING TO PARAMETERIZE THE ENVIRONMENTAL RESPONSE OF RNA SEQUENCE LIBRARIES AND USE COMPUTATIONAL APPROACHES EVALUATE HOW THESE ENVIRONMENTAL RESPONSES CAN LIMIT OR PROMOTE RNA ADAPTATIONS UNDER DIFFERENT EVOLUTIONARY SCENARIOS SUCH AS FLUCTUATING ENVIRONMENTS. THIS RESEARCH WILL ADVANCE OUR ABILITY TO CRITICALLY EVALUATE THE CAPACITY OF RNA CENTERED LIFE TO ADAPT TO NEW AND CHANGING ENVIRONMENTS. METHODOLOGY AS A MODEL SYSTEM WE WILL USE GROUP I RIBOZYMES. THESE RIBOZYME CAN CATALYZE A REVERSE-SPLICING REACTION THAT RESULTS IN A SUBSTRATE SEQUENCE BECOMING ATTACHED TO ITS 3 END. WE WILL USE THIS ATTACHED SEQUENCE TO SELECT MOLECULES THAT HAVE REACTED. BY COUNTING THE POPULATION FREQUENCY OF A SPECIFIC SEQUENCE BEFORE AND AFTER SELECTION WE WILL CALCULATE RIBOZYME FITNESS. FOR OUR MODEL ENVIRONMENTAL GRADIENT WE WILL USE MAGNESIUM ION CONCENTRATION. GROUP I RIBOZYMES SHOW A SIGMOIDAL RESPONSE TO INCREASING MG2+ CONCENTRATION THAT CAN BE FIT BY THE HILL EQUATION. DIFFERENT VARIANTS SHOW MAGNESIUM DEPENDENT RESPONSES THAT DIFFER IN BOTH THE INFLECTION POINT AND THE HILL COEFFICIENT BUT PREVIOUS WORK HAS BEEN LIMITED TO A FEW SEQUENCES. WE WILL CONSTRUCT A LIBRARY OF SEQUENCE NEIGHBORS AND DETERMINE THE RELATIVE ACTIVITY OF EACH SEQUENCE UNDER 10 DIFFERENT MAGNESIUM CONCENTRATIONS THAT COVER THE EXPECTED RESPONSE CURVES. FOR EACH SEQUENCE VARIANT WE WILL FIT THE MG2+ RESPONSE CURVE TO THE HILL EQUATION AND EXTRACT THE FIT PARAMETERS. WE WILL THEN MAP THESE PARAMETERS BACK TO SEQUENCE SPACE AND DETERMINE THE CHANGE IN MAGNESIUM DEPENDENCE CAUSED BY CHANGES IN THE NUCLEOTIDE SEQUENCE. WE DEVELOP EVOLUTIONARY SIMULATIONS TO PREDICT HOW POPULATIONS OF RNA MOLECULES WOULD ADAPT TO CONSTANT OR FLUCTUATING MAGNESIUM LEVELS. RELEVANCE THE PROPOSED RESEARCH WILL HELP UNDERSTAND THE ORIGIN EVOLUTION DISTRIBUTION AND FUTURE OF LIFE IN THE UNIVERSE WHICH IS THE GOAL OF THE NASA EXOBIOLOGY PROGRAM (APPENDIX C.5). RNA MOLECULES ARE BELIEVED TO PLAY A CENTRAL ROLE IN THE ORIGINS AND EARLY EVOLUTION OF LIFE. AS SUCH THE PROPOSED EXPERIMENTS WILL HELP ELUCIDATE THE ABILITY OF RNA CENTERED LIFE TO ADAPT TO NEW ENVIRONMENTS OR ENVIRONMENTAL FLUCTUATIONS. THE RIBOZYMES STUDIED ARE USED AS A MODEL OF MOLECULES THAT MAY HAVE CONTRIBUTED TO THE ORIGINS AND EARLY EVOLUTION OF LIFE. IN TURN THE RESEARCH WILL ALSO HELP UNDERSTAND HOW THE ENVIRONMENT CAN CONSTRAIN AND PROMOTE THE ABILITY OF RNA CENTERED LIFE TO ADAPT.