Transverse Magnetic Tweezers Allowing Coincident Epifluorescence Microscopy on Horizontally Extended DNA.
Longitudinal magnetic tweezers (L-MT) have seen wide-scale adoption as the tool-of-choice for stretching and twisting a single DNA molecule. They are also used to probe topological changes in DNA as a result of protein binding and enzymatic activity. However, in the longitudinal configuration, the DNA molecule is extended perpendicular to the imaging plane. As a result, it is only possible to infer biological activity from the motion of the tethered superparamagnetic microsphere. Described here is a "transverse" magnetic tweezers (T-MT) geometry featuring simultaneous control of DNA extension and spatially coincident video-rate epifluorescence imaging. Unlike in L-MT, DNA tethers in T-MT are extended parallel to the imaging plane between two micron-sized spheres, and importantly protein targets on the DNA can be localized using fluorescent nanoparticles. The T-MT can manipulate a long DNA construct at molecular extensions approaching the contour length defined by B-DNA helical geometry, and the measured entropic elasticity agrees with the worm-like chain model (force < 35 pN). By incorporating a torsionally constrained DNA tether, the T-MT would allow both the relative extension and twist of the tether to be manipulated, while viewing far-red emitting fluorophore-labeled targets. This T-MT design has the potential to enable the study of DNA binding and remodeling processes under conditions of constant force and defined torsional stress.
Department of Biology, University of York, Heslington, York, YO10 5DD, UK.
Stephen J
SJ
Cross
Department of Biology, University of York, Heslington, York, YO10 5DD, UK.
Claire E
CE
Brown
Department of Biology, University of York, Heslington, York, YO10 5DD, UK. christoph.baumann@york.ac.uk.
Christoph G
CG
Baumann
D004247:DNA / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D060328:Magnetic Phenomena; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D036103:Nanotechnology / Q000295:instrumentation; D052898:Optical Tweezers; D000072760:Single Molecule Imaging / Q000295:instrumentation*
D004247:DNA
Coincident fluorescence microscopy; DNA micromanipulation; Single-molecule manipulation; Transverse magnetic tweezers