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-蛋白质结合通常导致DNA拓扑结构的全局变化，例如弯曲或扭结。为了使DNA弯曲，需要在限定双链体构象的结构单元中进行调整。整体DNA构象由许多因素决定，其中之一是核糖环的“皱褶”偏好。当简单核苷酸的呋喃糖环在南（S）糖皱褶（2 ′-内切，B DNA样）和北（N）糖皱褶（3 ′-内切，A DNA/RNA样）之间处于动态平衡时，在掺入DNA链时，呋喃糖环采用优选的构象。在典型的B样DNA双链体中，参与拓扑调整（如弯曲）的碱基对呈现出改变的、更像A（N）糖的皱褶。DNA双链体的预排列更接近于结合状态（“弯曲”构象）可以增加结合亲和力或降低与目的蛋白质的解离能。如项目Z 01 BC 006174中所述，已对基于双环[3.1.0]己烷模板系统的独特合成核苷酸类似物的制备进行了改进，并研究了单体的构象。这种修饰的支架可以根据[3.1.0]支架上碱基的相对位置将糖皱褶锁定在N或S构象。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'），一种原型B型DNA。在上一份年度报告中，我们已经完成了一项NMR研究，使用残留偶极偶联（RDC）对DNA的纳入N-锁定胸苷，并表明这些修改导致弯曲的双链体取决于性质和数量的修改添加。我们还从单原子中子散射（SANS）实验中获得了初步证据，即当N-锁胸苷被构建到DNA链中时，DNA确实会发生弯曲，这项工作正在继续进行其他修饰的DNA。我们已经重复了这些低聚物的生物物理研究，严格审查了相应的N-锁定腺嘌呤取代的低聚物，并检查了6个DNA的一个或两个S-锁定胸苷或腺嘌呤掺入圆二色性（CD），差示扫描量热法（DSC）和核磁共振（NMR）。两个N-锁腺嘌呤衍生物的熔化温度高于天然的十二聚体，这表明通过添加A-样单体的矛盾稳定。然而，我们的断言，S-锁定单体（B-样DNA构象）的预组织将更有效地促进组装和稳定的双链体是不正确的，基于完整的生物物理数据集，我们现在已经为所有六个se修饰的DNA的。通过DSC、NMR和CD测量，我们发现在大多数衍生物中存在浓度依赖性的第二不稳定物种的形成。通过紫外（UV）和DSC测量的熔融数据是互补的，并且证实了这一结果。我们已经分配了这些衍生物的NMR光谱，并正在进行基于我们新开发的方法的RDC的测量。此外，我们已经组装的N-锁胸苷类似物的距离数据表，这些正在进行分析，并与通过广泛的分子建模研究获得的数据进行比较。在上一份报告中提到的用特定的13 C标记重建结构单元以提高NMR实验灵敏度的想法尚未实现，但仍然是研究长期计划的一部分。