This proposal describes experiments to isolate novel nucleic acid catalysts, RNA and DNA esterases, from pools of random sequence molecules. An in vitro genetic selection will be used to purify molecules that can bind to phosphonate transition state analogues (TSAs). These analogues mimic transitory intermediates in ester hydrolysis and have proven to be excellent targets for the generation of catalytic antibodies. Specific targets for in vitro selection experiments will include TSAs that have successfully elicited catalytic antibodies, and phosphonates coupled to peptides. A number of methods are proposed to improve the nascent technology of in vitro selection; the goal of these technical advances will be to allow the selection of nucleic acids that can bind ligands from pools of up to 10(15) different sequences in only a few days. The proposed research is directly relevant to understanding nucleic acid structure and chemistry. New RNA and DNA structures may be discovered that expand the repertoire seen in natural nucleic acids. Extant RNA enzymes (ribozymes) catalyze only one class of reactions, phosphodiester bond transfers, and nucleic acid esterases would allow the chemistry of an entirely new set of active sites to be studied. The generation of nucleic acid esterases through in vitro selection experiments would be a first step towards the creation of molecules whose activities could be tailored to virtually any reaction or substrate. This work may have important medical applications because nucleic acids, unlike catalytic antibodies, can be introduced into and function inside of cells. Selection experiments with peptides and peptide-phosphonates should provide insights into the chemical basis of nucleic acid-protein interactions; results from these experiments can aid in understanding the mechanisms of gene and metabolic regulation.
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