NMR Group Project: Biophysical Studies of Oligonucleotid

DNA-蛋白质结合通常会导致DNA拓扑结构的全局变化，如弯曲或扭结。为了使DNA弯曲，需要在定义双相构象的结构单元中进行调整。DNA的整体构象是由许多因素决定的，其中之一是核糖环的“折叠”偏好。简单核苷酸的呋喃糖环在南(S)糖环（2'-endo， B DNA样）和北(N)糖环（3'-endo， a DNA/ rna样）之间处于动态平衡状态，当被纳入DNA链时，呋喃糖环采用首选构象。在典型的b样DNA双工中，涉及拓扑调整（如弯曲）的碱基对呈现出改变的，更像a样(N)的糖褶。DNA双链的预先排列更接近于结合状态（“弯曲”构象）可能增加结合亲和力或降低感兴趣蛋白质的解离能。如项目Z01 BC 006174所述，对基于双环[3.1.0]己烷模板体系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。根据碱基在[3.1.0]支架上的相对位置，这种修饰的支架可以将糖袋锁定为N或S构象。将修饰的N-胸腺嘧啶和N-腺嘌呤核苷酸插入到Dickerson Drew十二聚体（5'- cgcgaattcgcg -3'）中，这是一种典型的b型DNA。在上一份年度报告中，我们已经完成了一项核磁共振研究，利用DNA上含有n锁胸苷的残余偶极偶联（rdc），并表明这些修饰会导致双链的弯曲，这取决于添加修饰的性质和数量。我们还从单原子中子散射（SANS）实验中获得了初步证据，当n -锁定胸苷被植入DNA链时，DNA确实会弯曲，这项工作将继续用于其他修饰的DNA。我们重复了这些低聚物的生物物理研究，严格检查了相应的n -锁定腺嘌呤取代低聚物，并通过圆二色性（CD），差示扫描量热法（DSC）和核磁共振（NMR）检查了6个具有s -锁定胸腺嘧啶或腺嘌呤结合的DNA。两种n锁定腺嘌呤衍生物的熔化温度高于天然十二聚体，这表明通过添加类a单体实现了矛盾的稳定。然而，基于我们目前对所有6个se修饰DNA的完整生物物理数据集，我们关于s锁定单体（b样DNA构象）的预组织将更有效地促进双链的组装和稳定的断言是不正确的。我们发现，通过DSC、NMR和CD测量，在大多数这些衍生物中形成了第二种不稳定物质的浓度依赖性形成。紫外（UV）和DSC测量的熔体数据是互补的，证实了这一结果。我们已经分配了这些衍生物的核磁共振光谱，并根据我们新开发的方法进行了rdc的测量。此外，我们还汇编了n锁胸苷类似物的距离数据表，并将这些数据与广泛的分子模拟研究获得的数据进行分析和比较。在上一份报告中提到的用特定的13C标记重新制作构建块以提高核磁共振实验灵敏度的想法尚未实现，但仍然是研究的长期计划的一部分。

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.