Cation-induced folding dynamics of the human telomeric G4 in 25 mM KCl. (A) Schematic representation of the folding of hTelo into various G4 conformations. Green and red circles placed at the G4 structures indicate the Cy3 and Cy5 fluorophores, respectively. (B) Representative FRET trajectory of single G4 molecule revealing several E states observed upon G4 folding. Colored solid lines indicate the FRET efficiency of these states as obtained from the HMM analysis. The black dashed line indicates the time of K+ addition. (C) Transition density plot showing the probability of transitions between different E states. (D and E) Dwell time histogram of the E≈0.3 and E≈0.73 states fitted with a single- and double-exponential functions, respectively (black lines). (F) Representative transfer efficiency histograms of G4 prior addition and for different time periods after addition of 25 mM K+, showing the slow kinetics of G4 folding and distribution of several E states. The last histogram was obtained for a sample that was thermally annealed and equilibrated for 24 h. Histograms were fitted globally to a mix of four Gaussian peak functions with shared peak positions and widths for each state (black lines). The color code used for identification of the different states and underlying FRET distributions is the same as in Figure 1B.