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底座在[3.1.0]支架上的相对位置将糖折叠锁定在N构象或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。在上一份年度报告中，我们完成了一项核磁共振研究，使用DNA上的残留偶极偶联(RDC)结合N-锁定胸苷，并表明这些修饰会导致双链的弯曲，这取决于添加的修饰的性质和数量。我们也有来自单原子中子散射(SANS)实验的初步证据，表明当N-锁定的胸苷被构建到链中时，DNA确实会弯曲，这项工作正在继续其他修饰的DNA。我们重复了对这些低聚体的生物物理研究，严格检查了相应的N-锁定腺嘌呤取代寡聚体，并通过圆二色谱(CD)、差示扫描量热法(DSC)和核磁共振(NMR)检测了6个带有S锁定的胸腺嘧啶或腺嘌呤掺入的DNA。两个N-锁定腺嘌呤衍生物的熔融温度比天然十二聚体的熔融温度高，这表明A类单体的加入具有矛盾的稳定性。然而，基于我们已有的所有六种硒修饰的DNA的完整生物物理数据，我们认为用S锁定的单体(B-类DNA构象)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。我们通过差示扫描量热法、核磁共振和CD测量发现，在大多数这些衍生物中形成了浓度依赖的第二不稳定物种的形成。紫外(UV)和差示扫描量热(DSC)测量的熔体数据是互补的，并证实了这一结果。我们已经指定了这些衍生物的核磁共振谱，并正在根据我们新开发的方法进行RDC的测量。此外，我们还汇编了N-锁定胸苷类似物的距离数据表，这些数据表正在进行分析，并与通过广泛的分子模拟研究获得的数据进行比较。上一篇报告中提到的重建具有特定13C标记的积木以增强核磁共振实验灵敏度的想法尚未实现，但仍是该研究长期计划的一部分。

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.