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DNA Polymerase with Single-Molecule Resolution: Activity, Inhibition, and Drug Re

单分子分辨率的 DNA 聚合酶：活性、抑制和药物研究

基本信息

批准号：
8722576
负责人：
PHILIP COLLINS
金额：
$ 27.91万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8722576
关键词：
Address Antibiotics Antineoplastic Agents Architecture Bacteria Biology Biotechnology Cancerous Carbon Carbon Nanotubes Catalysis Cells DNA DNA Polymerase Inhibitor DNA-Directed DNA Polymerase Data Development Disease Dissection Drug resistance Effectiveness Electronics Enzyme Kinetics Enzymes Evolution Eye Fluorescence Resonance Energy Transfer Foundations Future Goals Hour Human Virus Individual Industry Kinetics Malignant Neoplasms Methods Modification Monitor Mutagenesis Mutation Pharmaceutical Preparations Polymerase Proteins Publishing RNA-Directed DNA Polymerase Reagent Research Resolution Retroviridae Specificity Speed Structure Techniques Technology Therapeutic Therapeutic Studies Time Transistors Variant Viral Virus Work base cancer cell design drug discovery enzyme mechanism human DNA improved inhibitor/antagonist insight leukemia microorganism multidrug resistance inhibition therapy nanoscale novel novel strategies pathogen phosphodiester polymerization programs public health relevance research study response single molecule therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Viruses, cancer cells, and other pathogens rely on DNA polymerization to divide and propagate. More benignly, control over DNA polymerization helped establish and remains central to the biotechnology industry. The first goal of the proposed research is the development of nanoscale electronic circuits with single DNA polymerase molecules wired into a carbon nanotube-based field effect transistor. This architecture provides an exquisitely sensitive method for uncovering the kinetics, dynamics and individual steps required for enzymatic catalysis. The long-term goal of this research is to apply DNA polymerase nanocircuits to examine the enzyme's mechanism along with inventing new methods to investigate drug discovery and drug resistance. The experiments proposed here apply a new tool for single-molecule studies to focus on DNA polymerase, a long-studied enzyme with an extensive toolbox of approaches and reagents for its study. Single molecule studies of DNA polymerase using FRET also provide a firm foundation for the proposed experiments. Tethering individual molecules of DNA polymerase to carbon-based nanocircuits allows observation of the enzyme catalyzing phosphodiester bond formation with unprecedented time resolution (single microseconds) and duration (up to hours). Unexplored with the precision of single molecule studies, mutations to DNA polymerase conferring drug resistance have been identified for both anti-cancer and anti-viral treatments. Understanding how such mutations allow the enzyme to avoid inhibition, yet remain functional, could guide the development of more effective anti-cancer and anti-viral compounds.

描述（由申请人提供）：病毒、癌细胞和其他病原体依赖于DNA聚合来分裂和繁殖。更有利的是，对DNA聚合的控制帮助建立了生物技术产业，并一直是该产业的核心。这项研究的第一个目标是开发纳米级电子电路，将单个DNA聚合酶分子连接到基于碳纳米管的场效应晶体管中。这种结构提供了一种非常灵敏的方法，用于揭示酶催化所需的动力学、动力学和各个步骤。这项研究的长期目标是应用DNA聚合酶纳米电路来研究酶的机制沿着发明新的方法来研究药物发现和耐药性。这里提出的实验应用了一种新的单分子研究工具，专注于DNA聚合酶，这是一种长期研究的酶，具有广泛的研究方法和试剂工具箱。利用FRET对DNA聚合酶的单分子研究也为所提出的实验提供了坚实的基础。将DNA聚合酶的单个分子拴在碳基纳米电路上，可以以前所未有的时间分辨率（单微秒）和持续时间（长达数小时）观察催化磷酸二酯键形成的酶。未经探索的单分子研究的精度，突变的DNA聚合酶赋予耐药性已被确定为抗癌和抗病毒治疗。了解这些突变如何使酶避免抑制，但仍保持功能，可以指导更有效的抗癌和抗病毒化合物的开发。