SOLID STATE NMR STUDIES OF DNA AND RNA DYNAMICS

长期以来，人们一直认为， 为了充分理解双链DNA 与转录因子相互作用，并在一般情况下执行完整的 它的生物学功能。 因此，我们认为， DNA的结构经过多年的发展，高分辨率 用X射线衍射和溶液核磁共振方法获得DNA结构模型 显示来自规范B形式的序列特异性变化。 是 合理地问，如何一个同样详细的图片的内部 DNA的动力学将改变我们对这些结构变异的看法， 从而使我们理解DNA如何发挥其生物学功能。 固体核磁共振是研究局域动力学的理想技术 生物聚合物从近溶液状态到结晶状态 环境 此外，一些新的固态NMR技术 近年来，人们已经开发出了一种新的方法， 生物分子结构的高精度和精确度 广泛的样品条件范围，没有分子量限制。 在 在未来的五年里，我们计划使用固态核磁共振结构和 动态技术来定义结构之间的关系， DNA和RNA的动力学和功能。 具体来说，我们建议 以下五个阶段的计划：1）使用固态NMR来探索 DNA寡聚体中局部动力学的序列特异性， 限制酶、转录因子和 甲基转移酶; 2）使用固态NMR来研究甲基转移酶的动力学。 DNA甲基化的结构影响，特别是在富含CpG的DNA中 序列; 3）使用固态NMR技术研究动力学 和锤头状和发夹状的催化位点的结构 核酶; 4）继续开发和优化固态NMR 高分子量核酸结构解析技术 acids.将这些NMR技术扩展到17.6特斯拉， 多共振CPMAS探针的构造，从而有助于 完成具体目标1-4; 5）合成所有同位素标记的 实现特定目标1-4所需的DNA和RNA亚磷酰胺。

It has been long accepted that detailed structural information is necessary in order to fully understand the means by which duplex DNA interacts with transcription factors, and in general performs the full range of its biological functions. Accordingly, our view of the structure of DNA has evolved through the years, as high resolution structural models of DNA obtained by X-ray diffraction and solution NMR display sequence specific variations from the canonical B form. It is reasonable to ask how an equally detailed picture of the internal dynamics of DNA would modify our view of these structural variations and thus our understanding of how DNA performs its biological functions. Solid state NMR is an ideal technique for probing the localized dynamics of biopolymers from near solution conditions to the crystalline environment. In addition, a number of novel solid state NMR techniques have been developed in recent years that enable the definition of biomolecular structure with high accuracy and precision over the same broad range of sample conditions, with no molecular weight limit. In the next five years we plan to use solid state NMR structural and dynamical techniques to define the relationship(s) between structure, dynamics and function in DNA and RNA. Specifically we propose the following five stage program: 1) Use solid state NMR to explore the sequence specificity of localized dynamics in DNA oligomers containing binding sites for restriction enzymes, transcription factors and methyltransferases; 2) Use solid state NMR to investigate the dynamic and structural impact of DNA methylation, especially in CpG-rich DNA sequences; 3) Use solid state NMR techniques to investigate the dynamics and structure of the catalytic sites of the hammerhead and hairpin ribozymes; 4) Continue to develop and optimize solid state NMR techniques for structure elucidation in high molecular weight nucleic acids. Extend these NMR techniques to 17.6 Tesla by completing construction of a multi-resonant CPMAS probe, thus aiding in the completion of specific goals 1-4; 5) Synthesize all isotopically labeled DNA and RNA phosphoramidites required to achieve specific goals 1-4.