King, GA;
Spakman, D;
Peterman, EJG;
Wuite, GJL;
(2022)
Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers.
Optical Tweezers : Methods in Molecular Biology book series
, 2478
pp. 243-272.
10.1007/978-1-0716-2229-2_9.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1007/978-1-0716-2229-2_9 |
| Publisher version: | https://doi.org/10.1007/978-1-0716-2229-2_9 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | DNA structure, DNA supercoiling, DNA topology, Microfluidics, Optical tweezers, DNA, DNA Topoisomerases, Type I, DNA, Superhelical, Nanotechnology, Optical Tweezers |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10174428 |
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