Chapter title |
Transverse Magnetic Tweezers Allowing Coincident Epifluorescence Microscopy on Horizontally Extended DNA.
|
---|---|
Chapter number | 7 |
Book title |
Chromosome Architecture
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3631-1_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3629-8, 978-1-4939-3631-1
|
Authors |
Stephen J. Cross, Claire E. Brown, Christoph G. Baumann |
Editors |
Mark C. Leake |
Abstract |
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. |
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