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-蛋白差异通常会导致DNA拓扑的全球变化，例如弯曲或扭结。为了使DNA弯曲，需要在定义双链体构象的结构单元中进行调整。总体DNA构象由许多因素定义，其中之一是核糖环的“冰球”偏好。虽然简单核苷酸的呋喃糖环处于南（S）糖冰球（2'-endo，b dna样）和北（N）糖冰球（3'-endo，DNA/RNA样）之间的动态平衡，并在掺入DNA链中后，呋喃糖环采用了富烷糖环。在典型的B样DNA双链体中，诸如弯曲之类的拓扑调节的碱基对假设发生了变化的，更类似A的（N）糖冰可。 DNA双链体对结合状态（“弯曲”构象）的预先安排可能会增加结合亲和力或减少与感兴趣蛋白质的分离能量。如Project Z01 BC 006174中所述，基于Bicyclo 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完善，并研究了所研究的单体的构象。这种修饰的脚手架可以将糖冰球锁定在n或s构象中，具体取决于基部在3.1.0脚手架上的相对位置。将修饰的N-胸苷和N-腺苷核苷酸插入迪克森Drew DodeCamer（5'-CGCGAATTCGCG-3'），一种原型B型DNA。通过圆形二色性，差异扫描量热法和NMR获得的生物物理数据为改性糖单位对DNA结构的影响提供了证据。在上次的年度报告中，我们指出，NMR化学移位分配以及百合会的全面热力学和CD数据，其中胸苷被锁定的N模拟替代。我们还在新程序的背景下分析了残留的偶性偶联（RDC），以快速评估已经知道高分辨率结构的低分辨率结构的低聚物中的弯曲。我们在800 MHz的NMR研究阐明了我们的原始数据，然后用于分析三个T取代寡核苷酸的弯曲。我们表明，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替代影响的残基的数据来定义DNA弯曲。这将大大缩短分析替代DNA低聚物的全球变化的分析时间。此外，我们通过CD和NMR光谱检查了相应的腺嘌呤取代的低聚物。最初的数据表明，这些低聚物实际上相对于天然十二焦点是稳定的。这与将核苷酸预构建为B样（2'-endo）构象的想法是一致的，它将更有效地促进双链体的组装。与俄亥俄州立大学的助理教授贾斯汀·吴（Justin Wu）一起，我们开始通过分析完整的RDC列表来充分描述替代寡聚的全球折叠。我们目前正在设计一种新的合成程序，以准备锁定的N和S构建块，并具有特定的13C标记，以增强NMR实验的灵敏度。