A Comprehensive Structural and Dynamic Map of DNA Duplex Ends

描述（由申请人提供）：DNA双端结构和动力学可以影响许多酶依赖的过程，如噬菌体Mu的转位，以及HIV dsDNA拷贝整合到目标染色体DNA中。尽管它们在生物学中至关重要，但DNA双端在核磁共振和晶体结构研究中的代表性明显不足，并且它们的动态特性尚未直接测量。该项目的长期目标是建立一个全面的DNA双工端动态和结构图谱。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个独特序列）。我们将使用我们实验室开发的用于单分子测量的仪器来生成地图偶联核磁共振波谱。该仪器基于由细菌毒素-溶血素形成的纳米级孔。当单个DNA发夹被外加电场捕获在这个孔中时，双链茎就悬浮在孔前庭中。初步结果表明，DNA捕获过程中的低频（kHz）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在Hz到MHz范围内提供DNA双端结构和动力学的无偏读取。我们将解决的具体问题包括：1)孔前庭和外加电场在多大程度上影响DNA动力学？2)双端动力学是否与发夹环身份和双杆长度无关？3)广泛的共识是，一些序列是异常刚性的（如A束），而另一些序列是高度柔性的（如TATA）。这些序列会导致纳米孔电流特征像预测的那样聚集在一起吗？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，因为结果可能是负面的，也就是说，我们可能会发现观察到的电流动态仅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，报告精确的动力学数据，这是适合于高通量实验的。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.