NMR Group Project: Biophysical Studies of Oligonucleotid

NMR 小组项目：寡核苷酸的生物物理研究

• 批准号: 7053872
• 负责人: Joseph John Barchi
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7053872
• 关键词: DNA binding protein; bioimaging /biomedical imaging; calorimetry; chemical substitution; circular dichroism; conformation; monomer; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleotide analog; nucleotides; protein structure; ribose; structural biology

DNA-protein biding often results in global changes in the DNA topology, such as bending or kinking. For DNA to bend, there needs to be adjustments in the structural units that define the duplex conformation. The overall DNA conformation is defined by many factors, one of which is the "pucker" preference of the ribose ring. While the furanose ring of a simple nucleotide is in dynamic equilibrium between a South (S) sugar pucker (2'-endo, B DNA-like) and a North (N) sugar pucker (3'-endo, A DNA/RNA-like), upon incorporation into a DNA strand, the furanose ring adopts a preferred conformation. In a typical B-like DNA duplex, the base pairs involved in a topological adjustment such as a bend assume an altered, more A-like (N) sugar pucker. Prearrangement of the DNA duplex to more closely resemble the bound state ("bent" conformation) may increase the binding affinity or decrease the disassociation energy from a protein of interest. As outlined in project Z01 BC 006174, the preparation of unique synthetic nucleotide analogues based on a bicyclo 3.1.0 hexane template system has been refined and the conformation of the monomers studied. This modified scaffold can lock the sugar pucker in either an N or S conformation depending on the relative position of the base on the 3.1.0 scaffold. Modified N- thymidine and N-adenine nucleotides were inserted into the Dickerson Drew dodecamer (5'-CGCGAATTCGCG-3'), a prototypical B-type DNA. Biophysical data obtained through circular dichroism, differential scanning calorimetry, and NMR have provided evidence for the effects that the modified sugar unit(s) had on the DNA structure. In the last annual report, we had stated that both NMR chemical shift assignments and comprehensive thermodynamic and CD data for the oligomers where the thymidines were replaced by a locked N analogue were complete. We have also analyzed the residual dipolar coupling (RDC) in the context of a new procedure to rapidly assess bending in an oligomer where a high resolution structure is already known. Our NMR studies at 800 MHz clarified our original data and were then used in the analysis of the bending of the three T-substituted oligonucleotides. We showed that bending of the duplex progressively increases with the number and position of the substituted residues. This technique has the potential to define DNA bending by comparing data of residues that are not affected by the substitution. This will dramatically shorten the analysis time for the resolution of global changes of substituted DNA oligomers. In addition, we have examined the corresponding adenine-substituted oligomers by CD and NMR spectroscopies. Initial data suggest that these oligomers actually are stabilized relative to the native dodecamer. This would be consistent with the idea that preorganization of the nucleotides into a B-like (2'-endo) conformation would more efficiently facilitate assembly of the duplex. Along with assistant professor Justin Wu at the Ohio State University, we are starting to fully characterize the global folds of the substituted oligomers through the analysis of a full list of RDCs. We are currently devising a new synthetic procedure to prepare the locked N and S building blocks with specific 13C labeling for enhanced sensitivity in the NMR experiments.

DNA-蛋白质结合通常会导致 DNA 拓扑结构发生整体变化，例如弯曲或扭结。为了使 DNA 弯曲，需要对定义双链体构象的结构单元进行调整。总体 DNA 构象由许多因素决定，其中之一是核糖环的“褶皱”偏好。而简单核苷酸的呋喃糖环在南 (S) 糖褶皱（2'-endo，B DNA 样）和北 (N) 糖褶皱（3'-endo，A DNA/RNA-）之间处于动态平衡。类似），在掺入 DNA 链后，呋喃糖环采用优选构象。在典型的 B 样 DNA 双链体中，参与拓扑调整（例如弯曲）的碱基对呈现出改变的、更像 A (N) 的糖褶皱。 DNA 双链体的预排列使其更接近结合状态（“弯曲”构象）可以增加结合亲和力或降低目标蛋白质的解离能。正如项目 Z01 BC 006174 中所述，基于双环 3.1.0 己烷模板系统的独特合成核苷酸类似物的制备已得到改进，并对单体的构象进行了研究。这种修饰的支架可以将糖褶皱锁定在 N 或 S 构象，具体取决于碱基在 3.1.0 支架上的相对位置。将修饰的 N-胸苷和 N-腺嘌呤核苷酸插入 Dickerson Drew 十二聚体 (5'-CGCGAATTCGCG-3')（一种典型的 B 型 DNA）中。通过圆二色性、差示扫描量热法和 NMR 获得的生物物理数据为修饰糖单元对 DNA 结构的影响提供了证据。在上一份年度报告中，我们指出，胸苷被锁定的 N 类似物取代的低聚物的 NMR 化学位移归属以及综合热力学和 CD 数据均已完成。我们还在新程序的背景下分析了残余偶极耦合（RDC），以快速评估已知高分辨率结构的低聚物的弯曲度。我们在 800 MHz 下的 NMR 研究澄清了我们的原始数据，然后用于分析三种 T 取代寡核苷酸的弯曲。我们发现双链体的弯曲随着取代残基的数量和位置逐渐增加。该技术有可能通过比较不受取代影响的残基数据来定义 DNA 弯曲。这将大大缩短解决取代 DNA 寡聚物全局变化的分析时间。此外，我们还通过 CD 和 NMR 光谱检查了相应的腺嘌呤取代低聚物。初始数据表明这些寡聚体实际上相对于天然十二聚体是稳定的。这与将核苷酸预组织成 B 样（2'-endo）构象将更有效地促进双链体组装的想法是一致的。我们与俄亥俄州立大学的助理教授 Justin Wu 一起，开始通过分析完整的 RDC 列表来全面表征取代低聚物的全局折叠。我们目前正在设计一种新的合成程序，以制备具有特定 13C 标记的锁定 N 和 S 结构单元，以提高 NMR 实验的灵敏度。