TY - JOUR
T1 - Transverse magnetic tweezers allowing coincident epi-fluorescence microscopy on horizontally extended DNA
AU - Cross, Stephen John
AU - Brown, Claire E.
AU - Baumann, Christoph George
N1 - © 2016, Springer Science+Business Media New York. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details.
PY - 2016/6/10
Y1 - 2016/6/10
N2 - 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 epi-fluorescence 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 localised 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-labelled targets. This T-MT design has the potential to enable the study of DNA binding and remodelling processes under conditions of constant force and defined torsional stress.
AB - 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 epi-fluorescence 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 localised 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-labelled targets. This T-MT design has the potential to enable the study of DNA binding and remodelling processes under conditions of constant force and defined torsional stress.
U2 - 10.1007/978-1-4939-3631-1_7
DO - 10.1007/978-1-4939-3631-1_7
M3 - Article
SN - 1940-6029
VL - 1431
SP - 73
EP - 90
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -