Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping
2019
Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors, to the mechanical response of soft materials within the cell. Yet in the extreme-force regime, limitations in current approaches have restricted biomolecular studies, particularly under conditions of constant-force and when combined with single-molecule fluorescence. We have met these challenges with a surface-free force spectroscopy approach based on high-speed single-molecule hydrodynamic trapping, which is not only inexpensive and accessible, but also able to probe extremely high tensions. Furthermore, our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a recently discovered hyperstretched (HS) state in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. The HS state of dsDNA has twice the contour length of B-DNA and was initially discovered under conditions of high tension in the presence of free intercalating dyes. It was hypothesized that regions of HS DNA could also be induced without the aid of intercalators if high-enough forces were applied, but this hypothesis had not been tested until now. Combining force application with single-molecule fluorescence imaging was critical for distinguishing HS DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that will be a significant addition to the single-molecule toolbox, enabling the mechanics of nanostructures to be probed in previously difficult to access regimes.
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