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碱基在3.1.0支架上的相对位置将糖皱褶锁定在N或S构象中。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。通过圆二色谱、差示扫描量热法和NMR获得的生物物理数据为修饰的糖单元对DNA结构的影响提供了证据。在上一份年度报告中，我们已经指出，两个NMR化学位移分配和全面的热力学和CD数据的低聚物，其中胸苷被取代的锁定N类似物是完整的。我们还分析了残余偶极耦合（RDC）的上下文中的一个新的程序，以快速评估弯曲的低聚物中的高分辨率结构是已知的。我们在800 MHz的NMR研究澄清了我们的原始数据，然后用于分析三个T-取代的寡核苷酸的弯曲。我们发现，弯曲的双链体逐渐增加的取代残基的数量和位置。该技术具有通过比较不受取代影响的残基的数据来定义DNA弯曲的潜力。这将大大缩短用于解析取代的DNA寡聚体的全局变化的分析时间。此外，我们还研究了相应的腺嘌呤取代的低聚物的CD和NMR光谱。初始数据表明，这些低聚物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 '-内切）构象将更有效地促进双链体的组装的想法一致。沿着俄亥俄州州立大学的助理教授Justin Wu，我们开始通过分析完整的RDC列表来充分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方法来制备具有特定13 C标记的锁定N和S构建块，以增强NMR实验中的灵敏度。