Fig. 4: Single-molecule analysis of three-color FRET data.

Experiments were performed on two-state DNA origami structures that were labeled with Atto647N, Cy3B and Atto488. While Cy3B is attached to a flexible tether, Atto647N and Atto488 are positioned close to the top (12 o’clock; state 1) and bottom (6 o’clock; state 2) binding sites, respectively. a Zoom-in of the L-shaped DNA origami structure and corresponding kinetic scheme. b Representative single-molecule intensity and FRET trajectories for binding sites with 7.5 nt overhang after alternating red-yellow-blue laser excitation. First panel: Intensities after blue excitation. Second panel: intensities after yellow and red excitation. Third panel: corresponding three-color FRET efficiencies. Fourth and fifth panels: Deep-LASI output for state transition and trace classification. B: blue; Y: yellow; R: red; ex: excitation; det: detection. c TDPs of the apparent FRET efficiency states reveal an apparent distance change for all three FRET pairs (BY (left), BR (middle), and YR channel (right) with dwell times of 1.75 s and 2.69 s for the upper and lower binding site, respectively, nearly identical to the two-color DNA origami structures (Fig. 3c). Total number of transitions: 5,013. d Frame-wise weighted state-wise apparent (gray) and corrected (color) smFRET efficiency histograms of the BY (left), BR (middle), and YR (right) FRET pairs. As expected, the accurate FRET efficiency of the BR pair is static (E = 0.36). As the position of Cy3B changes from state 1 to state 2, the accurate FRET efficiency changes from 0.36 to 0.81 (BY pair) and from 0.81 to 0.08 (YR pair). e Upper panel: Representative three-color smFRET traces for binding sites with 6.5 nt (7 nt with 1 nt mismatch), 7 nt and 8 nt overhangs after alternating RYB laser excitation. Bottom Panel: The corresponding state determined by Deep-LASI. f Extracted dwell times from mono-exponential fits for the lower (blue) and upper positions (black) for 6.5 nt (\({\tau }_{1}\): 0.31 s, \({\tau }_{2}\): 0.4 s), 7 nt (\({\tau }_{1}\): 0.66 s, \({\tau }_{2}\): 1.05 s), 7.5 nt (\({\tau }_{1}\): 1.75 s, \({\tau }_{2}\): 2.69 s) and 8 nt overhangs (\({\tau }_{1}\): 6.41 s, \({\tau }_{2}\): 9.54 s) (see Supplementary Figure 6.1 for more details). nt: nucleotides. Source data are provided as a Source Data file.