A Comprehensive Structural and Dynamic Map of DNA Duplex Ends

描述（由申请人提供）：DNA双链体末端的结构和动力学可影响许多酶依赖性过程，如噬菌体Mu的转座和HIV dsDNA拷贝整合到靶染色体DNA中。尽管它们在生物学中至关重要，但DNA双链体末端在NMR和晶体结构研究中的代表性显着不足，并且它们的动力学性质尚未直接测量。该项目的长期目标是建立一个全面的DNA双链末端的动态和结构图。该图谱将具有接近埃的精度，并且将包括DNA螺旋的最后六个位置的所有可能的碱基对组合（即46 = 4096个独特序列）。我们将使用的方法来生成映射耦合NMR光谱与仪器在我们的实验室开发的单分子测量。这种仪器是基于细菌毒素形成的纳米级孔，溶血素当单个DNA发夹被施加的电场捕获在该孔中时，双链体茎悬浮在孔前庭中。初步结果表明，低频（kHz）的电流噪声在DNA捕获过程中所造成的螺旋末端的结构变化。该R-21提案的目的是确定纳米孔检测器是否在Hz至MHz范围内给出DNA双链体末端结构和动力学的无偏读数。具体的问题，我们将解决包括：1）在何种程度上的孔前庭和所施加的电场影响DNA动力学？2)双链体末端动力学是否独立于发夹环身份和双链体茎长度？3)有一个广泛的共识，一些序列是异常刚性（如A束），而其他的是高度灵活的（如TATA）。这些序列是否会导致纳米孔电流信号如预测的那样聚集在一起？4)模式识别算法可以用于自动化数据分析吗？我们认为这项研究适合R-21资助，因为存在结果为负面的重大风险，即我们可能会发现观察到的电流动力学仅与纳米级蛋白质腔中捕获的DNA发夹的有限情况相关。然而，如果这项研究成功，它将产生一种观察DNA动力学的新方法，可以报告精确的动力学数据，并且可以进行高通量实验。这些纳米孔实验将用于指导后续的NMR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.