Fig. 3: Single-molecule analysis of two-color FRET data.

Experiments were performed with DNA origami structures exhibiting two FRET states. a Zoom-in of an L-shaped DNA origami structure labeled with Atto647N and Cy3B and corresponding kinetic scheme. The donor is attached to the flexible tether with a 7.5 nt overhang between the pointer and two single-stranded binding sites. FRET is expected between a high FRET state 1 (12 o’clock) and a low FRET state 2 (6 o’clock) interconverting at rates k₁₂ and k₂₁. b Representative single-molecule and apparent FRET trace after alternating red-yellow (RY) laser excitation. Deep-LASI classifies the trace and determines the underlying state for each frame. D: donor; A: Acceptor; ex: excitation; det: detection. c TDPs determined using Deep-LASI (left) and HMM (right) are shown revealing two interconverting states with apparent FRET values of 0.8 and 0.2. The two states are labeled in white. Total number of transitions: n_Deep-LASI = 15,958, n_HMM = 21,243. d CDFs extracted from the TDPs shown in (c) and mono-exponential fits yield dwell times of 1.76 s and 2.64 s, respectively. The errors in the dwell times are the 95% confidence intervals returned by the fitting procedure (estimated from the Jacobian matrix). e A comparison of the cumulative dwell-time distribution determined using Deep-LASI - HMM for τ₁ (gray) and τ₂ (cyan). f Histograms of trace-wise determined correction factors for direct excitation, crosstalk and detection efficiency, either derived automatically by Deep-LASI (gray histograms, median in black) or determined manually (blue lines, median in cyan) (see Supplementary Note 5). g Apparent (left) and corrected (right) frame-wise smFRET efficiency histograms for 1499 dynamic traces from a total of 6100 traces. The states have corrected peak FRET efficiencies of 0.07 and 0.81. The histograms from traces selected by Deep-LASI are shown in gray and by manual selection in blue. Source data are provided as a Source Data file.