This proposal addresses the need for new reagents targeted toward specific sequences of DNA and RNA. Such reagents would have potential use in both diagnostic and therapeutic applications. The reagents described (in the proposal) are peptide nucleic acid (PNA) oligomers, which are synthetic analogs of DNA and RNA in which the sugar-phosphodiester backbone is replaced by a polyamide. PNAs hybridize to complementary single-stranded DNA and RNA with high affinity and sequence selectivity. Most applications of PNA will require hybridization to a target sequence that is part of a folded structure that could impose significant barriers to binding by sterically blocking access to the target site. PNA can bind to duplex DNA by strand invasion but only certain sequences can be targeted. The growing awareness of the importance on nonduplex DNA secondary structures and the rich variety of secondary and tertiary structures exhibited by RNA have prompted us to initiate studies on the ability of PNA to target these structures, both in simple model systems and within the context of a large, folded RNA molecule. We will use a variety of spectroscopic, calorimetric and gel electrophoretic methods to study the thermodynamics and kinetic factors controlling PNA hybridization to these targets. The principal aim of this proposal is to broadly examine the scope of PNA hybridization to structured targets to better evaluate the potential utility of PNA in diagnostic and therapeutic applications. The following Specific Aims will allow us to achieve this goal: * Examine binding of PNA to DNA and RNA hairpin motifs. * Study binding of homopyrimidine PNA to single- and double-stranded RNA sequences * Evaluate PNA-DNA and PNA-RNA hybridization kinetics. * Effect of RNA tertiary structure on PNA hybridization.
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