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Force determination in lateral magnetic tweezers combined with TIRF microscopy

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF)...

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Autores principales: Madariaga-Marcos, J., Hormeño, S., Pastrana, C. L., Fisher, G. L. M., Dillingham, M. S., Moreno-Herrero, F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royal Society of Chemistry 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831119/
https://www.ncbi.nlm.nih.gov/pubmed/29461549
http://dx.doi.org/10.1039/c7nr07344e
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author Madariaga-Marcos, J.
Hormeño, S.
Pastrana, C. L.
Fisher, G. L. M.
Dillingham, M. S.
Moreno-Herrero, F.
author_facet Madariaga-Marcos, J.
Hormeño, S.
Pastrana, C. L.
Fisher, G. L. M.
Dillingham, M. S.
Moreno-Herrero, F.
author_sort Madariaga-Marcos, J.
collection PubMed
description Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)–TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.
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spelling pubmed-58311192018-03-14 Force determination in lateral magnetic tweezers combined with TIRF microscopy Madariaga-Marcos, J. Hormeño, S. Pastrana, C. L. Fisher, G. L. M. Dillingham, M. S. Moreno-Herrero, F. Nanoscale Chemistry Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)–TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation. Royal Society of Chemistry 2018-03-07 2018-02-20 /pmc/articles/PMC5831119/ /pubmed/29461549 http://dx.doi.org/10.1039/c7nr07344e Text en This journal is © The Royal Society of Chemistry 2018 http://creativecommons.org/licenses/by/3.0/ This article is freely available. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence (CC BY 3.0)
spellingShingle Chemistry
Madariaga-Marcos, J.
Hormeño, S.
Pastrana, C. L.
Fisher, G. L. M.
Dillingham, M. S.
Moreno-Herrero, F.
Force determination in lateral magnetic tweezers combined with TIRF microscopy
title Force determination in lateral magnetic tweezers combined with TIRF microscopy
title_full Force determination in lateral magnetic tweezers combined with TIRF microscopy
title_fullStr Force determination in lateral magnetic tweezers combined with TIRF microscopy
title_full_unstemmed Force determination in lateral magnetic tweezers combined with TIRF microscopy
title_short Force determination in lateral magnetic tweezers combined with TIRF microscopy
title_sort force determination in lateral magnetic tweezers combined with tirf microscopy
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831119/
https://www.ncbi.nlm.nih.gov/pubmed/29461549
http://dx.doi.org/10.1039/c7nr07344e
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