Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion.
Single-Molecule Angular Optical Trapping for Studying Transcription Under Torsion.
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DOI：
10.1007/978-1-4939-8556-2_16
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发表时间：
2018
影响因子：
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通讯作者：
Jie Ma;Chuang Tan;Michelle D. Wang
中科院分区：
文献类型：
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作者：
Jie Ma;Chuang Tan;Michelle D. Wang
光镊是一种灵活而强大的单分子工具，在生物物理研究中得到了广泛的应用。凭借其拉伸和扭曲DNA的能力，并同时测量其力和扭矩，它们为获得对蛋白质马达和蛋白质-DNA相互作用功能的新见解提供了绝佳的机会。最近，一种新的DNA超螺旋检测使用角光镊（AOT）已被开发用于研究扭矩的产生在转录过程中。在这里，我们提供了一个详细而实用的指南来执行此技术。以细菌RNA聚合酶（RNAP）为例，我们提出了构建和校准AOT仪器、制备DNA模板以及获取和分析DNA超螺旋转录实时数据的方案。虽然这些协议最初是用E. coliRNAP，他们可以很容易地适应研究其他基于DNA的马达蛋白。
Optical tweezers are flexible and powerful single-molecule tools that have been extensively utilized in biophysical studies. With their ability to stretch and twist DNA, and measure its force and torque simultaneously, they provide excellent opportunities to gain novel insights into the function of protein motors and protein-DNA interactions. Recently, a novel DNA supercoiling assay using an angular optical tweezers (AOT) has been developed to investigate torque generation during transcription. Here, we provide a detailed and practical guide to performing this technique. Using bacterial RNA polymerase (RNAP) as an example, we present protocols for constructing and calibrating an AOT instrument, preparing DNA templates, and acquiring and analyzing real-time data for transcription under DNA supercoiling. While these protocols were initially developed withE. coliRNAP, they can be readily adapted to study other DNA-based motor proteins.