Interestingly, our cation-induced folding experiments reveal that the chair (E≈0.88) is the preferred first populated short-lived folded conformation (80% of all trajectories showing real-time folding) (Figure 1B and Supplementary Figure S1A–D), an observation that is in excellent agreement with results from recent ensemble stop-flow experiments (21). In contrast to more complex structural arrangements necessary for the formation of other conformations, the folding into the chair conformation per se can occur through a simple bending of a G-hairpin (transiently populating the unfolded ensemble) (21,22), which may preferentially favor this conformational transition. In the remaining 20% of cases the 2-tetrad basket conformation forms first, which can be explained by the fact that for the formation of this conformation only one K+ cation is required. It should be noted, however, that we estimate similar energy barriers for all folding transitions (Supplementary Table S4). In addition, we find that the unfolding of the chair G4 is slower by approximately a factor of two (kU = 0.15 s−1) compared to the unfolding of the 2-tetrad basket and hybrid conformations. This observation implies kinetic trapping of the anti-parallel chair conformation (49) and can explain the prevalence of this conformation at the beginning of the folding process (Figure 1F).