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

我们建议应用一种二维固体核磁共振 PI最近发明的一种技术，用于测定 脱氧核糖核酸和与之相关的结合分子的结构。 这项技术使用魔角旋转来获得高精度的 不同取向DNA膜的分辨率核磁共振谱 纤维纺丝技术，同时对每个方向进行编码 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来给出取向 个体群体的分布函数：无先验 假设这些函数可以组合在一起以生成 分子结构。建议的技术具有一些 与现有结构物理方法相比的优势 测定，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，这些光谱可区分 DNA的主要构象之间，不同的 这些构象的结构模型，以及 结合药物分子相对于DNA的不同排列 纤维。我们建议首先建造一种装置，用于 定向天然DNA膜的可重复性生产 通过改变电解液可以产生的各种形式 浓度和环境湿度，并通过2D-核磁共振分析 这些异构体的结构，提纯和去除 现有模型中的模棱两可。然后，我们将研究 定义序列的合成DNA的构象，以及 将它们的结构与天然材料的结构进行比较。最后， 我们将合成一系列同位素标记的DNA结合 配体，其中包括几个临床上重要的细胞毒性和 抗病毒药物，并确定其络合物的结构 用高分子量的DNA。这将在很短的时间内成为可能 药物分子与DNA碱基对的比率较低。终极的 目标是建立一种新的物理化学工具来帮助 新型药理活性物质的表征与开发 材料，并增加对过程的理解 基因复制、转录和调控。

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.