nl7b04842_si_006.avi (1.5 MB)

Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence

Download (1.5 MB)
posted on 23.02.2018, 00:00 by Graeme A. King, Andreas S. Biebricher, Iddo Heller, Erwin J. G. Peterman, Gijs J. L. Wuite
The ability to measure mechanics and forces in biological nanostructures, such as DNA, proteins and cells, is of great importance as a means to analyze biomolecular systems. However, current force detection methods often require specialized instrumentation. Here, we present a novel and versatile method to quantify tension in molecular systems locally and in real time, using intercalated DNA fluorescence. This approach can report forces over a range of at least ∼0.5–65 pN with a resolution of 1–3 pN, using commercially available intercalating dyes and a general-purpose fluorescence microscope. We demonstrate that the method can be easily implemented to report double-stranded (ds)­DNA tension in any single-molecule assay that is compatible with fluorescence microscopy. This is particularly useful for multiplexed techniques, where measuring applied force in parallel is technically challenging. Moreover, tension measurements based on local dye binding offer the unique opportunity to determine how an applied force is distributed locally within biomolecular structures. Exploiting this, we apply our method to quantify the position-dependent force profile along the length of flow-stretched DNA and reveal that stretched and entwined DNA moleculesmimicking catenated DNA structures in vivodisplay transient DNA–DNA interactions. The method reported here has obvious and broad applications for the study of DNA and DNA–protein interactions. Additionally, we propose that it could be employed to measure forces in any system to which dsDNA can be tethered, for applications including protein unfolding, chromosome mechanics, cell motility, and DNA nanomachines.