Chapter title |
Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions
|
---|---|
Chapter number | 20 |
Book title |
Single Molecule Analysis
|
Published by |
Humana, New York, NY, October 2023
|
DOI | 10.1007/978-1-0716-3377-9_20 |
Pubmed ID | |
Book ISBNs |
978-1-07-163376-2, 978-1-07-163377-9
|
Authors |
Julene Madariaga-Marcos, Pierre Aldag, Dominik J Kauert, Ralf Seidel |
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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