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Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions

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Single Molecule Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2694))

Abstract

Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.

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Author information

Authors and Affiliations

  1. Molecular Biophysics Group, Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Leipzig, Germany

    Julene Madariaga-Marcos, Pierre Aldag, Dominik J. Kauert & Ralf Seidel

Authors
  1. Julene Madariaga-Marcos
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  2. Pierre Aldag
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  3. Dominik J. Kauert
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  4. Ralf Seidel
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Corresponding author

Correspondence to Ralf Seidel .

Editor information

Editors and Affiliations

  1. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    Iddo Heller

  2. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    David Dulin

  3. LaserLaB Amsterdam and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

    Erwin J.G. Peterman

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Madariaga-Marcos, J., Aldag, P., Kauert, D.J., Seidel, R. (2024). Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions. In: Heller, I., Dulin, D., Peterman, E.J. (eds) Single Molecule Analysis . Methods in Molecular Biology, vol 2694. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3377-9_20

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  • DOI: https://doi.org/10.1007/978-1-0716-3377-9_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3376-2

  • Online ISBN: 978-1-0716-3377-9

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