NMR OF HIV REVERSE TRANSCRIPTASE PRIMERS

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DESCRIPTION (Provided by the applicant): RNA hairpin oligonucleotides will be synthesized which contain the three modified nucleosides found in the anticodon domain of human tRNA LYS3, the primer for HIV reverse transcriptase. The solution structures of these RNA hairpins will be determined using NMR spectroscopy. The high-resolution structures will be used to understand how the modified nucleosides rncm5s2U, ms2t6A, and pseudouridine provide structural stability to the anticodon domain of tRNA and why this is important for proper function of tRNALYS3 as the reverse transcriptase primer. Binding studies between the modified RNA hairpins and HIV reverse transcriptase will be done to elucidate how modification affects reverse transcriptase recognition. NMR spectroscopy will be used to localize the divalent metal ion binding sites in E.coli and human tRNALYS3. Paramagnetic manganese relaxation studies and NOE measurements of cobalt hexanimine complexed with the tRNA hairpins will be used as analogs of the magnesium ions that we have shown bind specifically to the modified RNAs. The solution structure of the RNA complex formed between tRNA and the A-rich loop of HIV genomic RNA will be determined using heteronuclear multidimensional NMR. The fully modified anticodon domain of human tRNA will be used to form the RNA-RNA complexes in order to understand how the natural modifications contribute to structural stabilization and to provide a structural target for therapeutic development. NMR spectroscopy will be used to study the solution structure of the entire tRNA isoacceptor from E.coli in its fully modified form. These structural studies will elucidate the nature of the folded core of tRNALYS and the structural basis for reverse transcriptase recognition elements in the D stem and loop of tRNALYS