Combined Magneto-Optical Tweezers and Supperesolution Fluorescence Imaging for Probing Dynamic Single-Molecule Topology of DNA, and Protein Machines that Manipulate DNA Topology

Abstract

Conventional magnetic tweezers (MT) designs suffer from a few limitations: (i) it is not possible to independently control torque while maintaining the desired force magnitude. The applied torque is orders of magnitude higher than biologically relevant values, precluding direct measurement of torque. (ii) They do not image the biological molecule. Rather, they image the magnetic beads attached to the molecules and infer the transformation of the molecules. This limits experiments to only high-torque processes that result in measurable change of molecular geometry, and even in these experiments the location of events on the molecule cannot be known. (iii) Due to the video-imaging based position detection of the magnetic bead, as opposed to interferometry based tracking typically used in optical tweezers, the low spatial and temporal resolutions prevent the observation of many molecular activities.The above limitations have been overcome individually but there has not been a design that overcomes all the drawbacks. Here we propose magneto-optical tweezers (MOT) to independently apply torques and forces at biologically relevant values while also arranging the DNA molecules in a transverse orientation to allow fluorescence microscopy to be performed and utilising back-focal-plane interferometry for high resolution tracking [1, 2]. We also present preliminary data on DNA topological response to MOT manipulation.1. Miller, H., Zhou, Z., Wollman, A.J., and Leake, M.C. Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. Methods 2015.2. Zhou, Z., Miller, H., Wollman, A., and Leake, M. Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy. Photonics 2015. 2, 758.