Fig. 3: Integration of SpeedyTrack and smFRET for freely diffusing molecules.
a–f Results with a donor-acceptor dual-labeled DNA hairpin. For each timepoint, the exposure time was 800 µs and the vertical shift time was 12.5 µs for 25 rows. a An example SpeedyTrack streak in the donor and acceptor channels for a hairpin molecule freely diffusing in a Tris buffer (200 mM NaCl). b Schematic: Closing and opening of the hairpin give high and low FRET signals, respectively. c FRET efficiency time trace from (a), calculated for each timepoint as the detected single-molecule intensity in the acceptor channel divided by the sum intensity of both channels. d Reconstructed spatial trajectory, colored by the FRET value. e Time-correlated conditional probabilities for a molecule to stay in the high-FRET (closed) state in solutions containing different amounts of NaCl, generated from ~5000 SpeedyTrack smFRET time traces under each condition (Supplemental Table 2). Error bars: standard errors via bootstrapping. f Hairpin opening and closing rates, kopen and kclose, extracted from the correlation results in (e) by fitting to an exponential decay model (Methods). Error bars: 95% confidence intervals. g–h Results on the hybridization of a donor dye-tagged 8-base DNA strand with an acceptor dye-tagged complementary 100-base strand. Exposure time was 300 µs and vertical shift time was 7.5 µs for 15 rows for each timepoint. g Schematic: The short strand and the hybridized strand give low and high FRET signals, respectively. h Two-dimensional distribution of mean FRET efficiency vs. estimated diffusion coefficient for individual single-molecule trajectories longer than 12 timepoints. Insets: Schematics of the three resolved states, and example trajectories (307.5 µs timesteps) colored by the FRET value as in (d).
