NMR OF HIV REVERSE TRANSCRIPTASE PRIMERS

描述（由申请人提供）：RNA发夹寡核苷酸将被 合成的含有反密码子中发现的三个修饰的核苷 人类tRNA LYS3的结构域，HIV逆转录酶的引物。的 这些RNA发夹的溶液结构将使用NMR来确定 谱高分辨率结构将用于了解 修饰的核苷rncm5s2U、ms2t6A和假尿苷提供了结构上的 tRNA反密码子结构域的稳定性以及为什么这对正确的 tRNIS3作为逆转录酶引物的功能。结合研究 在修饰的RNA发夹和艾滋病毒逆转录酶之间进行， 阐明修饰如何影响逆转录酶识别。 NMR光谱将用于定位二价金属离子结合位点 在大肠杆菌和人tRNATIS3中。顺磁锰弛豫研究和NOE 将使用与tRNA发夹复合的钴己亚胺的测量 作为镁离子的类似物，我们已经表明， 修饰的RNA tRNA与富含A的RNA形成的RNA复合物的溶液结构 HIV基因组RNA的环将使用异质性多维 匪r人tRNA的完全修饰的反密码子结构域将用于形成 为了理解自然修饰是如何 有助于结构稳定，并提供一个结构目标， 治疗发展 NMR光谱将用于研究整个溶液的结构。 来自大肠杆菌的完全修饰形式的tRNA同功受体。这些结构 研究将阐明tRNATIS折叠核心的性质， D茎中逆转录酶识别元件的结构基础 和tRNATIONS的循环

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