A Comprehensive Structural and Dynamic Map of DNA Duplex Ends

DNA 双链体末端的全面结构和动态图

• 批准号: 7024252
• 负责人: MARK A AKESON
• 金额: $ 17.28万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7024252
• 关键词: DNA; artificial intelligence; bioengineering /biomedical engineering; biomedical equipment; chemical kinetics; computational biology; computer program /software; computer system design /evaluation; electric field; method development; molecular dynamics; nanotechnology; nuclear magnetic resonance spectroscopy; nucleic acid structure; nucleobase; protein structure; technology /technique development

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.

描述（由申请人提供）：DNA双链末端的结构和动力学会影响众多酶依赖性过程，例如噬菌体MU的换位，以及H​​IV DSDNA拷贝的整合到靶标染色体DNA中。尽管在生物学中至关重要，但在NMR和晶体结构研究中，DNA双链末端的代表性显着不足，并且尚未直接测量其动态特性。该项目的长期目标是建立DNA双链末端的全面动态和结构图。该地图将处于接近Angstrom的精度，并将包括DNA螺旋的最后六个位置的基本对的所有可能组合（即46 = 4096唯一序列）。我们将使用在实验室中开发的单分子测量的仪器来生成地图夫妇NMR光谱的方法。该仪器基于细菌毒素形成的纳米级孔，？ - 蛋白蛋白。当通过施加的电场在此孔中捕获单个DNA发夹时，双链茎悬挂在孔前庭中。初步结果表明，在DNA捕获过程中，低频（KHz）电流噪声是由螺旋末端的结构变化引起的。该R-21建议的目的是确定纳米孔检测器是否给出了HZ至MHz范围内DNA双链端结构和动力学的无偏读。我们将要解决的具体问题包括：1）孔隙前庭和应用电场在多大程度上影响DNA动力学？ 2）双链端动力学是否独立于发夹环的身份和双链茎长？ 3）有广泛的共识，某些序列异常刚性（例如A段），而另一些序列具有很高的灵活性（例如tata）。这些序列是否会像预测的那样引起纳米孔电流签名？ 4）模式识别算法可用于自动化数据分析吗？我们认为这项研究适合R-21资金，因为结果可能是负面的，即我们可能会发现观察到的当前动态仅与在纳米级蛋白腔中捕获的DNA发夹的有限情况有关。但是，如果研究成功，它将产生一种新的方法来观察DNA动力学，以报告精确的动力学数据，并且可以适合高通量实验。这些纳米孔实验将用于指导随后的NMR实验，反之亦然。