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Force determination in Lateral Magnetic Tweezers combined with TIRF microscopy

AutorMadariaga-Marcos, J.; Hormeño, S.; Pastrana, C.L.; Fisher, G.L.M.; Dillingham, M.S.; Moreno-Herrero, Fernando
Palabras claveMagnetic tweezers
Optical tweezers
Atomic force microscopy
TIRF microscopy
DNA molecules
Fecha de publicación2018
EditorRoyal Society of Chemistry (Great Britain)
CitaciónNanoscale
ResumenCombining 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, 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 the 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.
Versión del editorhttp://dx.doi.org/10.1039/c7nr07344e
URIhttp://hdl.handle.net/10261/160269
DOI10.1039/c7nr07344e
ISSN2040-3364
E-ISSN2040-3372
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