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首页> 外文学位 >Electronic control of DNA polymerase binding and unbinding to single DNA molecules tethered in a nanopore.
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Electronic control of DNA polymerase binding and unbinding to single DNA molecules tethered in a nanopore.

机译：电子控制DNA聚合酶与纳米孔中束缚的单个DNA分子结合和不结合。

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摘要

This work combines computer control with a nanopore sensor to detect and manipulate single DNA molecules and DNA/enzyme complexes captured in the pore. In the setup, a membrane protein self-assembles into a lipid bilayer and an electric potential applied across the membrane creates a measurable ionic current that flows through the pore. A sufficiently large potential produces an electric field capable of capturing DNA molecules and enzyme-bound DNA complexes in the pore. Captured unbound DNA molecules pass through the pore. Because enzymes are too large to enter the pore, enzyme bound to captured DNA must dissociate from the DNA before the DNA translocates through the pore. The basis for detection of translocating polynucleotides, such as DNA, is the measurable reduction in ionic current through the pore. Variations in the current amplitude can differentiate unbound DNA from enzyme-bound DNA in the pore and enable real-time detection of enzyme dissociation from captured DNA. A finite state machine automates detection and reaction to the dissociation of enzymes from captured DNA in real time. Moreover, additional characteristics of the current signal permit identification of the base present at the enzyme's catalytic site, providing a means for single-base sequencing. Combining these capabilities, this research explores methods for repeated control of a single tethered DNA molecule, suspended in the nanopore by voltage control and biochemical alteration. In the tethered configuration, the DNA is available for rapid and repeated binding of enzymes above the pore. The repeated binding and subsequent dissociation of enzymes represents a dramatic advance in the level of control of DNA in a nanopore. With such control of DNA, titration experiments allow investigation into the detectable limits of complex assembly, at the single-molecule level, with statistical significance and provide pre-steady-state biochemical information.
机译：这项工作将计算机控制与纳米孔传感器结合在一起，以检测和操纵单个DNA分子以及捕获在孔中的DNA /酶复合物。在该设置中，膜蛋白会自组装为脂质双层，并且跨膜施加的电势会产生可测量的离子电流，该电流流经孔。足够大的电势会产生能够捕获孔中DNA分子和酶结合的DNA复合物的电场。捕获的未结合的DNA分子穿过孔。由于酶太大而无法进入孔中，因此与捕获的DNA结合的酶必须先从DNA解离，然后DNA才能通过孔迁移。检测易位多核苷酸（例如DNA）的基础是通过孔的离子电流的可测量降低。电流幅度的变化可以使孔中未结合的DNA与酶结合的DNA区分，并能够实时检测酶与捕获的DNA的解离关系。有限状态机可实时自动检测和处理捕获的DNA中酶的解离。此外，电流信号的其他特征允许鉴定存在于酶催化位点的碱基，为单碱基测序提供了一种手段。结合这些功能，本研究探索了通过电压控制和生化改变对悬浮在纳米孔中的单个拴系DNA分子进行重复控制的方法。在栓系构型中，DNA可用于在孔上方快速且重复地结合酶。酶的重复结合和随后的解离代表了纳米孔中DNA控制水平的显着进步。通过这种对DNA的控制，滴定实验可以在单分子水平上研究复杂装配体的可检测限，具有统计意义，并提供稳态前的生化信息。

著录项

  • 作者

    Wilson, Noah A.;

  • 作者单位

    University of California, Santa Cruz.;

  • 授予单位 University of California, Santa Cruz.;
  • 学科 Biology Molecular.;Engineering Electronics and Electrical.;Biophysics General.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 106 p.
  • 总页数 106
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;生物物理学;无线电电子学、电信技术;
  • 关键词

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