MODIFIED NUCLEOSIDES AS TOOLS FOR MOLECULAR BIOLOGY

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The long-range goal of this research is to develop modified nucleosides suitable for applications that require either the introduction or the recognition of nucleic acid sequence degeneracy. The results from the initial period of this project support the hypothesis that azole carboxamides are able to function as natural base mimics in DNA recognition. Furthermore, the azole nitrogen substitution pattern has a profound influence on the base pairing specificity. We have sufficient information to rationally design a second generation of azole carboxamide nucleosides. Specific aims include: 1) Synthesis of a series of azole carboxamide nucleosides to investigate the role of DNA polymerase-nucleobase interactions in template directed synthesis. 2) Construction of second generation azole nucleobase containing oligonucleotides for biophysical characterization. 3) Investigation of oligonucleotides containing first and second generation azole nucleobases as templates for Taq DNA polymerase and investigation of azole nucleoside triphosphates as alternative substrates in PCR. 4) Exploration of two strategies for using the azole nucleobases in directed evolution technology. The analogs described in this proposal present a broad diversity of electronic configurations within the steric confines of natural nucleobase structure, thereby providing a unique opportunity to explore enzyme recognition and function. The techniques described in this proposal have the potential to provide a thorough exploration of diversity space in short random walks from a starting gene sequence. Although this proposal centers primarily around DNA polymerase, the interaction of azole nucleobase containing oligonucleotides with other nucleic acid enzymes is an unexplored territory with the potential to lead to both fundamental understanding of nucleic acid-protein interactions as well as the development of new tools for protein engineering and directed evolution.