NMR Group Project: Biophysical Studies of Oligonucleotid

DNA-蛋白质结合经常导致DNA拓扑结构的全局变化，如弯曲或扭曲。为了让DNA弯曲，需要对定义双链构象的结构单元进行调整。DNA的整体构象是由许多因素决定的，其中之一就是核糖环的“折叠”偏好。当一个简单核苷酸的呋喃糖环处于南方(S)糖折叠(2‘-endo，BDNA样)和北(N)糖折叠(3’-endo，ADNA/RNA样)之间的动态平衡时，当呋喃糖环进入DNA链时，呋喃糖环采用首选构象。在典型的B-类DNA双链中，参与拓扑调整(如弯曲)的碱基对假设一个改变的、更像A的(N)糖折叠。将DNA双链预先排列为更接近结合状态(“弯曲”构象)可能会增加结合亲和力或降低目标蛋白质的解离能。如项目Z01 BC 006174所述，基于双环3.1.0正己烷模板体系的独特的合成核苷酸类似物的制备已经得到改进，并研究了单体的构象。这种改进的支架可以根据3.1.0支架上碱基的相对位置将糖折叠锁定在N或S构象中。将修饰的N-胸苷和N-腺嘌呤核苷酸插入到典型的B型DNA-Dickerson Drew十二聚体(5‘-CGCGAATTCGCG-3’)中。通过圆二色谱、差示扫描量热法和核磁共振获得的生物物理数据已经为修饰的糖单元(S)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，胸苷被锁定的N类似物取代的低聚物的核磁共振化学位移指定和全面的热力学和CD数据都是完整的。我们还分析了残留偶极耦合(RDC)在一种新的程序，以快速评估低聚物中的弯曲已知的高分辨率结构。我们在800 MHz的核磁共振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，随着取代残基的数目和位置的增加，双链的弯曲程度逐渐增加。这项技术有可能通过比较不受取代影响的残基的数据来定义DNA弯曲。这将极大地缩短分析取代的DNA低聚体的全球变化的时间。此外，我们还用CD和核磁共振波谱研究了相应的腺嘌呤取代低聚物。初步数据表明，相对于天然十二聚体，这些低聚物实际上是稳定的。这与将核苷酸预先组织成类B(2‘-endo)构象将更有效地促进双链组装的想法是一致的。与俄亥俄州立大学的助理教授贾斯汀·吴一起，我们开始通过对RDC的完整清单的分析，全面描述取代寡聚体的全球折叠。我们目前正在设计一种新的合成工艺来制备具有特定13C标记的锁定的N和S构建块，以提高在核磁共振实验中的灵敏度。

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.