Analysis of Single DNA Polymerase Complexes at 5 Angstrom Precision in Real Time

描述(由申请人提供)： 项目概述本应用程序旨在实现对DNA聚合酶功能的电子控制，时间尺度与酶结合和催化的速度重叠。为了实现这一目标，我们将监测单个DNA聚合酶与纳米孔传感器在电压诱导张力下的相互作用。我们将表征在纳米孔中电压控制下捕获的聚合酶-DNA复合体的动力学、生化和结构特性。我们将优化聚合酶功能的纳米孔测量，带宽比使用传统技术可能的带宽高得多，并且允许在处理DNA时对数千种单个酶进行连续分析。我们相信这项研究是创新的，因为它将使用最近建立的纳米孔技术来识别和测量复制的各个催化周期中的易位步骤。应该能够区分聚合酶驱动的易位机制。这项工作与人类健康有关，因为DNA聚合酶错误结合核苷酸而产生的突变是癌症的根本原因。此外，纳米孔偶联聚合酶可以提供一种高速、低成本的基因组测序技术，几乎不会对环境造成影响。 公共卫生相关性： 本工作主要研究DNA聚合酶复制DNA的机制。它与人类健康有关，因为DNA聚合酶错误结合核苷酸而产生的突变是癌症的根本原因。此外，纳米孔偶联聚合酶可以提供一种高速、低成本的基因组测序技术，几乎不会对环境造成影响。

DESCRIPTION (provided by applicant): Project Summary This application aims to achieve electronic control of DNA polymerase function on a time scale that superimposes with rates of enzyme binding and catalysis. To achieve this aim, we will monitor the interaction of individual DNA polymerases with a nanopore sensor under voltage- induced tension. We will characterize kinetic, biochemical, and structural properties of polymerase-DNA complexes captured under voltage control in a nanopore. We will optimize nanopore measurements of polymerase function at significantly higher bandwidth than is possible using conventional techniques and in a manner that permits serial analysis of thousands of individual enzymes as they process DNA. We believe the study is innovative because it will employ a recently established nanopore technique to identify and measure translocation steps during individual catalytic cycles of replication. Discrimination between polymerase-driven translocation mechanisms should be achievable. This work is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact. PUBLIC HEALTH RELEVANCE: This work focuses on mechanisms of DNA replication by DNA polymerases. It is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact.