Flow chambers were prepared as previously described58 (link),59 (link). Briefly, quartz slides and coverslips were passivated with polyethylene glycol (5% biotinylated) and flow chambers constructed using double-sided sticky tape and sealed with epoxy. Pre-annealed dsDNA (final concentration 12.5 pM) was immobilized via biotin–streptavidin interactions. The flow chambers were imaged on a home-built, prism-based total internal reflection microscope with a 532-nm excitation laser (~2 mW), and images acquired on an EM-CCD camera (Andor) with a 30 ms exposure time. FRET efficiencies were calculated from integrated donor (ID) and acceptor (IA) intensities as FRET = IA/(ID + IA)54 (link),59 (link). Images and data were analyzed by custom IDL, MATLAB and R scripts (available upon request). FRET efficiency histograms were constructed by averaging the first ten frames of each trajectory, filtering traces which gave an average FRET value >1.2 or <-0.2 and binning with bins of 0.05. FRET states were determined by Gaussian fitting and reported as mean ± s.d. When two distributions of bound and unbound populations were observed, percentage values were calculated from the area under the fitted Gaussian of one population as a proportion of the total fitted area of both populations.