Institute of Structural and Molecular Biology, University College London, London, UK.
Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Methods Mol Biol. 2022;2478:243-272. doi: 10.1007/978-1-0716-2229-2_9.
Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between <5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced.
许多基因组过程导致 DNA 形成欠旋（负超螺旋）或过旋（正超螺旋）。这些 DNA 拓扑变化调节 DNA 结合蛋白的相互作用，包括转录因子、结构蛋白和拓扑异构酶。为了深入了解超螺旋 DNA 的结构和相互作用，我们最近开发了一种称为光学 DNA 超螺旋（ODS）的单分子方法。该方法使用标准的双阱光学镊子仪器快速产生负超螺旋 DNA（比非超螺旋 DNA 的螺旋扭曲度低 5%至 70%）。ODS 具有优势，因为它允许进行力谱学、荧光成像和超螺旋底物的空间控制的组合，这是大多数其他方法难以实现的。在这里，我们描述如何使用双阱光学镊子产生负超螺旋 DNA。为此，我们提供了关于合适 DNA 底物的设计和准备的详细说明，以及如何控制和校准所产生的超螺旋密度的分步指南。
