A Comprehensive Structural and Dynamic Map of DNA Duplex Ends

描述(申请人提供)：DNA双链末端的结构和动态可以影响许多酶依赖的过程，如噬菌体Mu的转座，以及HIV双链DNA拷贝整合到目标染色体DNA中。尽管DNA双链末端在生物学上至关重要，但在核磁共振和晶体结构研究中，它们的表达明显不足，而且它们的动态性质还没有被直接测量。该项目的长期目标是建立一个全面的DNA双链末端动态和结构图。该图谱将达到接近埃的精度，并将包括DNA螺旋最后六个位置的所有可能的碱基对组合(即46=4096个唯一序列)。我们将使用这种方法来生成MAP，将核磁共振波谱与我们实验室开发的单分子测量仪器结合起来。该仪器是基于细菌毒素？-溶血素形成的纳米级孔隙。当单个DNA发夹在这个毛孔中被外加电场捕获时，双链茎悬浮在毛孔前厅。初步结果表明，DNA捕获过程中的低频电流噪声是由螺旋末端的结构变化引起的。这项R-21提案的目的是确定纳米孔探测器是否能在赫兹到兆赫范围内无偏读取DNA双链末端结构和动力学。我们将解决的具体问题包括：1)毛孔前庭和外加电场在多大程度上影响DNA动力学？2)双链末端动力学是否独立于发夹环的同一性和双链茎长度？3)人们普遍认为，一些序列是异常刚性的(例如A束)，而另一些序列是高度灵活的(例如TATA)。这些序列是否会导致纳米孔电流信号像预测的那样聚集在一起？4)模式识别算法可以用于自动数据分析吗？我们认为这项研究适合R-21资助，因为结果是负的风险很大，即我们可能会发现，观察到的电流动态只与纳米级蛋白质腔中捕获的DNA发夹的有限情况有关。然而，如果研究成功，它将产生一种新的方法来观察DNA动力学，报告准确的动力学数据，并服从高通量实验。这些纳米孔实验将用于指导后续的核磁共振实验，反之亦然。

DESCRIPTION (provided by applicant): The structure and dynamics of DNA duplex ends can influence numerous enzyme-dependent processes such as transposition of phage Mu, and integration of HIV dsDNA copies into target chromosomal DNA. Despite their critical importance in biology, DNA duplex ends are significantly under-represented in NMR and crystal structure studies, and their dynamic properties have not been measured directly. The long-term goal of this project is to establish a comprehensive dynamic and structural map of DNA duplex ends. This map will be at near angstrom precision and will include all possible combinations of base pairs for the last six positions of the DNA helix (i.e. 46 = 4096 unique sequences). The approach we will use to generate the map couples NMR spectroscopy with an instrument developed in our laboratory for single molecule measurements. This instrument is based on a nanoscale pore formed by the bacterial toxin, ?-hemolysin. When individual DNA hairpins are captured in this pore by an applied electric field, the duplex stem is suspended in the pore vestibule. Preliminary results suggest that low frequency (kHz) current noise during DNA capture is caused by structural changes of the helix terminus. The aim of this R-21 proposal is to determine if the nanopore detector gives unbiased reads of DNA duplex end structure and dynamics in the Hz to MHz range. Specific questions we will address include: 1) To what extent does the pore vestibule and the applied electric field influence DNA dynamics? 2) Are the duplex end kinetics independent of hairpin loop identity and duplex stem length? 3) There is broad consensus that some sequences are unusually rigid (e.g. A tracts) while others are highly flexible (e.g. TATA). Do these sequences cause nanopore current signatures that cluster together as predicted? 4) Can pattern recognition algorithms be used to automate data analysis? We consider this research to be suited for R-21 funding because there is substantial risk that the outcome will be negative, i.e. we may find that the observed current dynamics are only relevant to the limited case of DNA hairpins captured in a nanoscale protein cavity. However, if the research is successful, it will yield a new way to observe DNA dynamics that reports precise kinetic data, and that is amenable to high throughput experiments. These nanopore experiments will be used to direct subsequent NMR experiments and vice versa.