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: 6944665
• 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/6944665
• 关键词: bioimaging /biomedical imaging; biophysics; chemical structure function; circular dichroism; conformation; intermolecular interaction; microcalorimetry; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleoside analog; nucleotide analog; phosphorylation; polynucleotides; structural biology

DNA-protein binding 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 a 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. We have fully assigned the NMR chemical shifts of the oligomers where either one or both thymidine base pairs were replaced by a locked N-thymidine. Both CD and calorimetric data indicate that stable duplexes are formed in all 3 oligomers and the melt temperatures of the modified DNA's are quite similar to the native duplex. However, there are perturbations close to the mutation site upon insertion of a "N" nucleoside in an otherwise "S" DNA duplex. These perturbations are highly localized and do not transmit along the DNA chain as has been reported for other conformationally-biased DNA constructs. In order to determine the degree of bending imparted by the modified base pairs, we are collaborating with Dr. Ad Bax and have performed NMR studies at 800 MHz which allowed us to modify our original assignments made from experiments at lower field. We have calculated residual dipolar coupling (RDC) constants from natural abundance 13C-1H data on the three oligomers and have compared these to those measured for the native dodecamer by the Bax group. We are currently calculating the structures of the modified DNA's based on the RDC measurements.

DNA-蛋白质结合经常导致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-腺嘌呤核苷酸插入到Dickerson Drew十二聚体（5 '-CGCGAATTCGCG-3'）（一种原型B型DNA）中。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。我们已经完全分配的NMR化学位移的寡聚体，其中一个或两个胸苷碱基对被替换为锁定的N-胸苷。CD和量热数据均表明，在所有3种寡聚体中形成稳定的双链体，并且修饰的DNA的解链温度与天然双链体非常相似。然而，在“S”DNA双链体中插入“N”核苷后，突变位点附近存在扰动。这些扰动是高度局部化的，并且不像已经报道的其他构象偏倚的DNA构建体那样沿DNA链沿着传递。为了确定修饰碱基对赋予的弯曲程度，我们与Ad Bax博士合作，在800 MHz下进行了核磁共振研究，这使我们能够修改根据较低场实验进行的原始任务。我们已经计算出残余偶极耦合（RDC）常数从自然丰度13 C-1H数据的三个低聚物，并将这些测量的天然十二聚体的Bax组。目前，我们正在计算基于RDC测量的修饰DNA的结构。