The observation of direct folded-to-unfolded transitions implies an extremely high degree of cooperativity of G4 folding/unfolding. Such level of cooperativity may be surprising, as previously several partially folded structures, such as G-hairpins and G-triplexes have been proposed to bridge the completely unfolded and folded G4 states, resulting in sequential folding pathways (21–24,26). These structures were also proposed to form during helicase-induced sequential unwinding of G4s (56,57). In our experiments with a G-triplex forming sequence such stable partially folded structures were not detected and are thus unlikely to contribute to any of the high E states here (Supplementary Figure S8). It should, however, be noted that we expect the unfolded state of G4s to be represented by a dynamic ensemble of a number of different unfolded and transiently formed partially folded conformations (29), possibly including G-hairpins and G-triplexes that may appear as a relatively broad peak at low transfer efficiencies in our single-molecule FRET histograms.