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: 7291782
• 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/7291782
• 关键词: NMR; Group; Project; Biophysical; Studies

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. In the last annual report, we had completed an NMR study using residual dipolar couplings (RDCs) on the DNA's incorporating N-locked thymidines and showed that these modifications caused bending of the duplex depending on the nature and number of modifications added. We also have preliminary evidence from single atom neutron scattering (SANS) experiments that the DNA does bend when N-locked thymidines are built into the strand and this work is continuing with other modified DNAs. We have repeated the biophysical studies on these oligomers, critically examined the corresponding N-locked adenine-substituted oligomers and examined six DNA's with either or both S-locked thymidines or adenine incorporation by circular dichroism (CD), Differential scaning calorimetry (DSC) and nuclear magnetic resonance (NMR). Melting temperatures for two of the N-locked adenine derivatives were higher that the native dodecamer suggesting a paradoxical stabilization by addition of A-like monomers. However, our assertion that preorganization with S-locked monomers (B-like DNA conformation) would more efficiently facilitate assembly of and stabilize the duplex was incorrect based on the complete set of biophysical data that we now have available for all six of the se modified DNA's. We found that there was a concentration-dependent formation of a second unstable species formed in most of these derivatives by DSC, NMR and CD measurements. Melt data by both ultraviolet (UV) and DSC measurements were complementary and confirmed this result. We have assigned the NMR spectra of these derivatives and are proceeding with the measurement of RDCs based on our newly developed method. In addition, we have assembled tables of the distance data for the N-locked thymidine analogues and these are being analyzed and compared with data obtained by extensive molecular modeling studies. The idea mentioned in the last report of remaking building blocks with specific 13C labeling for enhanced sensitivity in the NMR experiments has not been realized but is still part of the long range plan of the research.

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。在上一份年度报告中，我们使用残余偶极偶联 (RDC) 对 DNA 中掺入 N 锁胸苷的情况完成了一项 NMR 研究，结果表明，这些修饰会导致双链体弯曲，具体取决于所添加修饰的性质和数量。我们还从单原子中子散射 (SANS) 实验中获得了初步证据，表明当 N 锁胸苷构建到链中时，DNA 确实会弯曲，并且这项工作正在针对其他修饰的 DNA 继续进行。我们对这些寡聚物重复了生物物理学研究，严格检查了相应的 N 锁腺嘌呤取代寡聚物，并通过圆二色性 (CD)、差示扫描量热法 (DSC) 和检测了具有 S 锁胸苷或腺嘌呤掺入之一或两者的 6 个 DNA。核磁共振（NMR）。两种 N-锁腺嘌呤衍生物的熔解温度高于天然十二聚体，表明通过添加 A 类单体实现了矛盾的稳定化。然而，基于我们现在拥有的所有六种修饰体的完整生物物理数据集，我们关于使用 S 锁定单体（B 样 DNA 构象）进行预组织将更有效地促进双链体组装和稳定双链体的断言是错误的。 DNA 的。通过 DSC、NMR 和 CD 测量，我们发现大多数这些衍生物中存在浓度依赖性的第二种不稳定物质的形成。紫外 (UV) 和 DSC 测量的熔体数据是互补的，证实了这一结果。我们已经分配了这些衍生物的核磁共振谱，并正在根据我们新开发的方法进行 RDC 的测量。此外，我们还整理了 N 锁胸苷类似物的距离数据表，并且正在对这些数据进行分析，并与通过广泛的分子建模研究获得的数据进行比较。上一份报告中提到的用特定 13C 标记重新构建构建块以增强 NMR 实验灵敏度的想法尚未实现，但仍然是该研究长期计划的一部分。