The goal of this research, chosen by the BNP Panel from a palette of science presented in the previous submission, is """"""""to contribute to a fine-grained understanding of interactions"""""""" between polymerases (pols) and their substrates. We will: (1) synthesize nucleotide analogs that form Watson-Crick pairs with different hydrogen bonding patterns, C-glycosides, without an unshared electron pair (UEP) in the minor groove, with functionality in the major groove, with substantial contributions from minor tautomeric equilibria, and able to form purine-purine and pyrimidine-pyrimidine mismatches; (2) Mutate DNA pols and reverse transcriptases to develop enzymes better able handle unnatural nucleotides; and (3) Measure the ability of pols to incorporate these analogs to test hypotheses concerning their interaction between pols and their substrates. The technological goals are stated as a proposition: Should a biological chemist wish to put an unnatural nucleotide into a DNA molecule via template-directed polymerization, this research will identify the natural pol to do this best, suggest ways to mutate the pols to do it better, and suggest alternative forms of the nucleotide that are easier to incorporate. Three hypotheses will be tested: Hypothesis 1: Can answers to these questions predict how an unnatural nucleotide will interact with a pol? (a) Does the nucleotide have an unshared pair of electrons in the minor groove? (b) Is it a C-glycoside or an N-glycoside? (c) Does it present a large substituent to the major groove (d) What is the ratio of its tautomeric forms? (e) Can it form purine-purine and pyrimidine-pyrimidine mismatches joined by three hydrogen bonds? Hypothesis 2: Is the evolutionary history of a pol the best predictor of how it interacts with unnatural nucleotides? Hypothesis 3: To modulate the interaction between a pol and a particular nucleotide, are amino acids in the second and third """"""""Shell,"""""""" 10-18 A away from the active site Mg, the relevant ones to change? This project fits our long term goal in organic chemistry: to develop an artificial genetic system based on an expanded genetic information systems (AEGIS), and our overall vision for the future of molecular science, where evolutionary theory from biology will be joined to structure theory from chemistry, joining the two principal traditions in science, natural history and the physical sciences, in a way that permits the power of each to contribute to the nation's biomedical research needs.
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