For single-molecule FRET experiments we used a DNA construct consisting of a duplex DNA terminated by a G4 forming overhang AGGG(TTAGGG)3, corresponding to the human telomeric sequence (hTelo) that mimics the natural telomeric DNA. The DNA construct was labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores for FRET detection (Figure 1A and Supplementary Table S1). Cation-induced folding of G4s and the subsequent conformational dynamics was monitored upon rapid addition of K+ (20 mM Tris–HCl and 25 mM KCl, pH 7.5), while imaging on the microscope (Figure 1B). The sample was prepared and initially imaged under non-folding conditions (20 mM Tris–HCl and 25 mM LiCl, pH 7.5) and an unfolded state with low transfer efficiency (E) state (E≈0.3) was initially populated. Upon addition of K+, we observe a rapid increase of transfer efficiency as a result of G4 formation (Figure 1B). Thereafter, the majority of FRET trajectories show multiple transitions between several E states, reflecting the highly dynamic nature of G4 folding (Figure 1B and C). Four distinct E states were identified through an HMM analysis of the FRET trajectories (Figure 1B and Supplementary Figure S1). The lowest E state (E≈0.3) represents the unfolded overhang and the three higher E states centered at E≈0.57, E≈0.73 and E≈0.88 correspond to different folded G4 conformations. These states are in dynamic interconversion with dwell times <10 s, except for the E≈0.73 state, which shows an additional long-lived subpopulation with a dwell time an order of magnitude larger (Figure 1D, E and Supplementary Figure S2). This long-lived state is also evident in FRET trajectories, where after initial conformational dynamics between several transient E states, G4s get stabilized in the long-lived E≈0.73 state (Figure 1B and Supplementary Figure S1). The long dwell time is also evidenced by an increased occurrence of static trajectories with E≈0.73 at later stages after folding initiation (Supplementary Figure S1). In addition to fast conformational dynamics, our single-molecule experiments allowed monitoring the G4 folding kinetics occurring on slower timescales (Figure 1F). Prior to the addition of K+, the G4 overhang is unfolded and appears as a single peak at low transfer efficiencies. After addition of K+ three high E peaks become apparent in the histograms. Interestingly, the distribution of these states varies notably within the first 60 minutes after folding initiation. At the early stages the E≈0.88 state is dominant, while after 60 min the E≈0.73 state becomes the major population. The FRET histogram obtained 60 minutes after KCl addition looks very similar to the one obtained in conditions where the population distribution has reached a complete equilibrium (Figure 1F, lowest panel). These results indicate that G4s reach thermodynamic equilibrium rather slowly, adopting the most stable (lowest energy) conformation within several hours after folding initiation in the presence of 25 mM KCl (Figure 1F).