TARGET PROMOTED ALKYLATION OF NUCLEIC ACIDS

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Research interest in DNA alkylation continues to be sustained by numerous issues concerning nucleic acid structure, toxicology, and pharmacology. The importance of alkylating agents in chemotherapy cannot be underestimated despite the serious complications associated with such treatment. Selective modification of DNA is generally ascribed to the intrinsic chemistry and binding properties of the modifying agent since DNA typically acts as a passive receptor and target of reaction. The potential for DNA to act as a catalyst and control its own modification has received limited attention despite substantial advances in the related field of RNA catalysis. This proposal is designed to reveal a new and perhaps general mechanism of reagent activation that should broaden our understanding of potential mutagenic, chemotherapeutic and diagnostic reactants. Previous studies have demonstrated that duplex DNA can activate silyl phenol derivatives for alkylation and cross-linking. These derivatives had originally been constructed for fluoride-dependent reaction but were found to react spontaneously after binding to target nucleotide sequences. The mechanism of this process remains to be discovered. The proposed investigations will begin by localizing the region of duplex DNA that is responsible for the activation process. The mechanism will then be defined in part by its functional group and conformational requirements. These in turn will be identified by examining the effect of nucleotide analogues incorporated into the region of activation. Finally, the silyl-based chemistry will be extended to new reagent design and application to enhance its future utility in vitro and in vivo. Accordingly, the efficiency and specificity of target modification will be examined in DNA, RNA and protein complexes as a method to help define biological structure and reactivity. The efficiency of our lead compounds will also be tested with a variety of cell lines and compared to alkylating agents that are used clinically.