To obtain further mechanistic insight into the folding of G4s we extracted the probability and rate constants for each individual transition observed in the course of G4 folding (Figure 1C, Supplementary Figure S3, Supplementary Tables S2 and 3). We find that the majority of transitions occur between an unfolded and the different folded conformations i.e. the interconversion between folded conformations proceeds via an unfolding step (Figure 1C). Direct transitions between folded conformations were also observed, but are significantly less probable (< 5%) (Supplementary Table S2). It should be noted that direct interconversion between folded G4s is structurally implausible and will require at least partial unfolding (14). Therefore, we believe that the small number of direct transitions observed in our experiments may involve a rapid complete or partial unfolding step that, however, remains hidden due to limited time resolution. We find that the chair, 2-tetrad basket and hybrid 2 conformations are rapidly formed short-lived folded states (kF = 0.19-0.21 s−1 and kU = 0.15-0.35 s−1) (Supplementary Table S3) that are extensively observed at the early stages of G4 folding (Figure 1F).