SOLID-STATE 2D-NMR INVESTIGATION OF DNA & ITS COMPLEXES

DNA 的固态 2D NMR 研究

• 批准号: 3466599
• 负责人: GERARD STANISLAUS HARBISON
• 金额: $ 9.17万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466599
• 关键词: DNA; DNA binding protein; antiviral agents; chemical binding; chemical bond; conformation; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid sequence; nucleic acid structure; nucleobase

We propose to apply a two-dimensional solid-state NMR technique, recently invented by the PI, to the determination of the structure of DNA and of bound molecules associated with it. The technique uses magic-angle spinning to acquire high- resolution NMR spectra of oriented DNA films produced by a fiber-spinning technique, while encoding the orientations of each group in the polymer as sideband intensities in the 2D spectra. These intensities can be directly used to give the orientational distribution function of individual groups: without prior assumptions these functions can be combined to generate the structure of the molecule. The proposed technique has some advantages over existing physical methods of structural determination, and because it probes molecular orientations rather than dimensions, is likely to be complementary to them. Preliminary calculations show that the spectra distinguish between the major conformations of DNA, between different models of the structure of these conformations, and between different alignments of bound drug molecules relative to the DNA fiber. We propose first of all to construct an apparatus for the reproducible production of films of oriented native DNA in the various forms which can be produced by altering electrolyte concentration and ambient humidity, and to analyses via 2D-NMR the structure of these conformers, refining and removing ambiguities in existing models. We shall then examine the conformation of synthetic DNAs of defined sequence, and compare their structures with those of native materials. Finally, we shall synthesize a series of isotopically-labelled DNA-binding ligands, among them several clinically important cytotoxic and antiviral drugs, and determine the structure of their complexes with high-molecular-weight DNA. This will be possible at very low ratios of drug molecules to DNA base pairs. The ultimate goal is to establish a new physico-chemical tool to aid in the characterization and development of novel pharmacological materials, and to add to the understanding of the processes of genetic replication, transcription and regulation.

我们建议应用二维固体核磁共振 技术，最近发明的PI，以确定 DNA和与之相关的结合分子的结构。 该技术使用魔角旋转来获得高- 定向DNA膜的NMR谱 纤维纺纱技术，同时编码每个方向 聚合物中的基团作为2D光谱中的边带强度。 这些强度可以直接用于给出取向 单个组的分布函数：无先验 假设这些功能可以组合以生成 分子的结构。 所提出的技术具有一些 与现有的物理方法相比， 决定，因为它探测分子取向 而不是维度，很可能是对它们的补充。 初步计算表明， DNA的主要构象之间，不同的 这些构象的结构模型，以及 结合的药物分子相对于DNA的不同排列 光纤 我们建议首先建造一个装置， 定向天然DNA膜的可重复生产 可以通过改变电解质产生的各种形式 浓度和环境湿度，并通过2D-NMR进行分析 这些构象异构体的结构， 现有模型的模糊性。 然后，我们将研究 确定序列的合成DNA的构象，和 将它们的结构与天然材料进行比较。 最后， 我们将合成一系列同位素标记的DNA结合 配体，其中有几个临床上重要的细胞毒性和 抗病毒药物，并确定其复合物的结构 高分子量的DNA 这将是可能的， 药物分子与DNA碱基对的比率低。 最终 目标是建立一种新的物理化学工具， 新型药理学的表征和开发 材料，并增加对过程的理解， 基因复制、转录和调控。