Guanine-rich DNA and RNA sequences can fold into structures consisting of stacked guanine tetrads. These quadruplex structures are believed to play important roles in several biological contexts, including repressing transcription of the c-MYC oncogene, controlling alternative splicing of the pre-mRNA for hTERT, the reverse transcriptase component of telomerase and regulating the activity of telomerase. Molecules that bind to these quadruplex structures can serve as probes of biological function and potential therapeutics. High affinity PNA probes will be synthesized and targeted to DNA and RNA G-quadruplex structures. What distinguishes this proposal from standard antigene and antisense approaches to regulating gene expression is the fact that the PNAs will have homologous, rather than complementary, sequences to their targets. This strategy is feasible because G-rich PNAs are known to form hybrid quadruplexes with homologous DNA and RNA sequences. Hybridization thermodynamics and kinetics will be measured for PNA-DNA and PNA-RNA quadruplex formation using a combination of optical spectroscopy and surface plasmon resonance experiments. An important component of this project will be synthesizing various PNA designs will to optimize affinity and selectivity. Once this is achieved, PNAs will be synthesized and used to test binding to three G quadruplexes (2 DNA and 1 RNA) modeled on biologically relevant targets. Finally, PNAs will be introduced into mammalian cell culture to study inhibition of c-MYC transcription, alteration of hTERT pre-mRNA splicing and inhibition of telomerase activity.
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