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褶皱锁定在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。我们已经完全归属了其中一个或两个胸苷碱基对被锁定的N-胸腺嘧啶核苷取代的低聚物的核磁共振化学位移。CD和量热数据表明，3种低聚物中都形成了稳定的双链，修饰后DNA的熔融温度与天然双链非常接近。然而，当“N”核苷插入另一个“S”DNA双链时，在突变部位附近有扰动。这些扰动是高度局部化的，并不像已报道的其他构象偏向DNA结构那样沿着DNA链传播。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进行了核磁共振研究，使我们能够修改最初在低场实验中的指定。我们从天然丰度13C-1H数据计算了三种低聚物的剩余偶极耦合(RDC)常数，并与Bax小组测得的天然十二聚体的RDC常数进行了比较。我们目前正在根据RDC的测量结果计算修饰后的DNA的结构。

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.