Abstract
Combining 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, its 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 a 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.
Highlights
In recent years, there has been increasing interest in combining force spectroscopy with fluorescence microscopy.[1,2] These combined setups, built upon magnetic tweezers (MT), optical tweezers (OT), and atomic force microscopy (AFM), are powerful tools permitting the manipulation of individual molecules at the same time they are visualized
DNA has been directly visualized with fluorescence microscopy using intercalating dyes during the mechanical disassembly of viruses by AFM,[3] and proteins involved in DNA repair have been directly observed while their mechanical action on the DNA was probed with OT.[4]
In vertical MT, a pair of permanent magnets aligned with the optical axis pull superparamagnetic beads tethered to the flat surface of a flow cell by DNA molecules (Fig. 1A)
Summary
There has been increasing interest in combining force spectroscopy with fluorescence microscopy.[1,2] These combined setups, built upon magnetic tweezers (MT), optical tweezers (OT), and atomic force microscopy (AFM), are powerful tools permitting the manipulation of individual molecules at the same time they are visualized. Magnetic tweezers permit the simultaneous tracking of several individual (non)torsionally constrained DNA molecules anchored on the surface of a flow cell, while a force is applied in a controlled manner.[17] On the other hand, TIRF microscopy exhibits a superior signal-to-noise ratio over other fluorescence-based techniques. One of the most widespread manners of visualizing fluorescent DNA molecules on a surface at the same time they are sensing a force is to stretch them under a continuous flow.[2,23,24] Note that the force applied to flowstretched DNA is not constant along the DNA molecule, being larger at the anchoring point than at the tip, and it is difficult to estimate.[25] Here, we argue that a way to have accurate control of the pulling force in combined systems with TIRF microscopy is by using lateral magnetic pulling. Our lateral pulling device was combined with TIRF microscopy This setup gives access to experiments where one can simultaneously visualize DNA binding proteins under controlled stretching forces. Our work provides a guide to implement lateral magnetic tweezers compatible with TIRF microscopy and a reference of the force magnitude that can be applied
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
