Dynamic Force-Fluorescence Spectroscopy of a DNA Holliday Junction Rev…

As mechanical parameters are being recognized as important determinants of many biological processes, single-molecule manipulation techniques have been continually evolving to break new records in their sensitivity and stability 1-3. However, one biologically relevant detection regime that has been out of reach is that of high spatial resolution under weak applied forces. We have therefore developed a new single-molecule technique, whereby mechanical manipulation via optical tweezers and observation of molecular motion via single-molecule FRET are combined without compromising their individual capabilities. Using this approach we have studied the conformational dynamics of the Holliday junction under applied stretching force, that has revealed the existence a hidden intermediate state along the reaction coordinate. The length of helical arms is shown to be an important factor determining force sensitivity of the Holliday junction. This suggests that the recombination process occurring in the cell could be extremely sensitive to sub-pico Newton forces that last only milliseconds.