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Methods Mol Biol. 2018;1703:139-152. doi: 10.1007/978-1-4939-7459-7_10.

Single-Molecule Magnetic Tweezer Analysis of Topoisomerases.

Author information

1
Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.
2
Department of Physics and Astronomy, Northwestern University, Evanston, IL, USA.
3
Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL, 60208, USA. a-mondragon@northwestern.edu.

Abstract

Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases: double-stranded DNA (dsDNA) tethers, "braided" (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions. Additionally, we discuss how to build flow cells for bright-field MT microscopy, as well as how to noncovalently attach anti-digoxigenin to the coverslip surface for tethering digoxigenin-labeled DNAs. Finally, we describe procedures for the identification of a suitable DNA substrate for MT study and data collection.

KEYWORDS:

Bright-field microscopy; Flow cell; Functionalized DNA; Magnetic tweezers; Noncovalent antibody attachment; Single-molecule; Topoisomerases

PMID:
29177739
PMCID:
PMC6643975
DOI:
10.1007/978-1-4939-7459-7_10
[Indexed for MEDLINE]
Free PMC Article

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