微纳体系中基于不同空间位阻效应的单分子DNA聚合酶反应动力学研究

The ability to monitor DNA polymerase reaction kinetics with single-molecule and single-nucleotide resolution is the cornerstone of research on the mechanism of enzyme and concepts of advanced single-molecule DNA sequencing. Decreasing the catalytic reaction rate of DNA polymerase with high enzyme activity is significant for clearly distinguishing and probing different steps of reaction kinetics. The relationship between the reaction activity and different steric hindrance effect of single DNA polymerase molecule will be studied at the single-nucleotide resolution based on the evanescent wave of zero mode waveguide in this project, and the object of decreasing the catalytic reaction rate of DNA polymerase with high enzyme activity is expected to achieve. The main research contents in this project are shown as follows: The methods and processes of preparing biotinylated DNA polymerase at the different site of molecule structure will be studied by theoretical modeling and genic mutation; Selective immobilization methods to position single molecule exclusively above the zero mode waveguide floor and increasing the DNA polymerase molecular occupancies in the zero mode waveguide array will be researched by selective chemical modification methods; The DNA polymerase reaction kinetics will be analysised via the single molecule fluorescence signs, and the relationship between the reaction kinetics and different steric hindrance effect will be concluded. The research contents and results in this project will provide theory and technique basis for single-molecule DNA sequencing and further studying the reaction mechanism of DNA polymerase.

从单分子及单碱基水平研究DNA聚合酶反应动力学已成为探讨DNA聚合酶机理及发展单分子DNA测序技术的重要基石。在保持DNA聚合酶活性的前提下降低其催化反应速率对于更加清晰方便地识别和理解酶分子反应动力学具有重要意义。本项目从调整单分子DNA聚合酶在固定化过程中活性位点朝向的不同入手，基于零模波导表面消逝波原理在单碱基水平研究不同空间位阻效应与单分子酶反应动力学的关系，拟通过优化固定位点实现在保持酶分子活性前提下降低其反应速率的目标。具体为：通过理论计算和基因突变技术研究DNA聚合酶分子不同空间位点的生物素化方法; 利用化学修饰方法在零模波导固定单分子DNA聚合酶，并研究提高单分子酶在阵列零模波导中占有率的技术方法; 通过表征零模波导中单分子荧光数据分析酶反应动力学，总结不同空间位阻效应与反应动力学的关系。本项目将为进一步研究DNA聚合酶分子反应机理及单分子DNA测序提供理论及技术基础。

零模波导作为一种实时获得单分子荧光信息的器件已经被成功应用于单分子测序，同时也为从单分子及单碱基水平研究 DNA 聚合酶反应动力学提供了一种新的方法。本项目（31200643）从DNA聚合酶与荧光基团修饰碱基的反应活性、零模波导器件的制备工艺、单分子成像、单分子在零模波导器件的占有率等几大方面开展了系统性研究。经过系统实验筛选出四种DNA聚合酶能够以中等或较高的效率与荧光基团标记在3'端磷酸键上的核苷酸进行反应；利用聚焦离子束技术制备出直径100nm，反应体积为仄升量级的零模波导器件。利用全内反射荧光显微镜对单分子荧光成像及单分子选择性修饰方案进行了验证。通过对零模波导器件进行选择性修饰将单分子在零模波导阵列的占有率提高至60%左右。本项目的研究为进一步利用零模波导器件研究单分子DNA聚合酶提供了技术基础，同时荧光基团标记在3'端磷酸键上的核苷酸与众多DNA聚合酶的反应效率结果为后期的单分子测序提供了意义重大的参考数据。