The experiments described in this proposal are designed to explore the evolution of novel ribozyme (RNA enzymes) from populations of random sequences, and thus to shed light on the origins of biological catalysis, a fundamental question in molecular biology.
The specific aims of this proposal are to characterize the distribution of ribozymes in sequence space, to study the evolution of novel catalysts starting from simple binding motifs, to begin to define the catalytic mechanisms used by newly evolved ribozymes, and to explore the range of biologically relevant chemical reactions that can be catalyzed by RNA. A large number of ligase ribozymes have been isolated by in vitro selection and evolution. These ribozymes will be characterized to learn about the diversity, activity and informational complexity of their catalytic domains. The relative abundances of ribozymes of different activities and complexities will be determined. The degree to which the catalytic properties of simple and complex ribozymes can be optimized by in vitro evolution will be measured. The effectiveness of step-wise strategies for the isolation of more complex ribozymes will also be studied. In vitro evolution has been used to evolve an ATP binding domain into a large number of polynucleotide kinase ribozymes. The abundance of kinases will be compared in pools of completely random sequence RNAs vs. biased pools constructed around pre-defined substrate binding sites. Questions concerning the catalytic mechanisms employed by newly isolated ribozymes will be addressed by studying substrate binding and reaction pathway control in one particular polynucleotide kinase ribozyme. Finally, attempts will be made to select for new ribozymes that carry out redox reactions, and reactions related to protein synthesis. The information that will result from these experiments is critical for our understanding of the molecular biology of the origin and early evolution of life in the RNA World, and for the evolution and design of new ribozymes with potential therapeutic applications.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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Massachusetts General Hospital
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