SYNTHESIS OF 15N LABELED RNA FRAGMENTS

说明：本提案的长期目标是发展和 展示合成和利用所需的合成策略 特定修饰或标记的RNA片段。 的分子 用15 N和13 C位点特异性标记可以提供独特且 对局部RNA结构（包括氢）的独立模型洞察 键合、质子化、水合、防止水合和堆积。 15 N特异性标记的核苷的效用将通过以下方式扩展： 将13 C引入两个嘌呤的C8位置和两个嘌呤的C2位置 腺嘌呤 一对嘌呤中只有一个与15 N偶联， 将允许区分其他类似的NMR共振。 此外，13 C可以提供关于碱基堆积的独立信息。 使用特异性15 N和13 C标记开发的RNA合成程序 将通过使它们适应亚磷酰胺而更广泛地使用 法 由于许多简单的RNA基序喜欢非规范碱基对， 单凸起和四环现在已经通过15 N NMR表征， 新的核磁共振工作将主要集中在更复杂的系统，如内部 环，大发夹环，假结，吻环，四环受体， 核酶和蛋白质-RNA复合物。 利用15 N NMR确定金属 离子与RNA的结合，是其结构和功能的一个关键方面， 探讨了

DESCRIPTION: The long-term goals of this proposal are to develop and demonstrate synthetic strategies necessary for the synthesis and utilization of specifically modified or labeled RNA fragments. Molecules that are site-specifically labeled with 15N and 13C can provide unique and model-independent insight into local RNA structure, including hydrogen bonding, protonation, hydration, protection from hydration and stacking. The utility of 15N specifically labeled nucleosides will be expanded by introducing 13C into the C8 position of both purines and the C2 position of adenine. The resulting coupling with 15N in only one of a pair of purines will allow otherwise similar NMR resonances to be distinguished. Furthermore, the 13C can provide independent information on base stacking. The procedures developed for RNA synthesis using specific 15N and 13C labels will be made more widely available by adapting them to the phosphoramidite method. Since many of the simpler RNA motifs like non-canonical base pairs, single bulges, and tetraloops have now been characterized by 15N NMR, the new NMR work will focus primarily on more complex systems, such as internal loops, large hairpin loops, pseudoknots, kissing loops, tetraloop receptors, ribozymes, and protein-RNA complexes. The use of 15N NMR to define metal ion binding to RNA, a crucial aspect of its structure and function, will be explored.