Next, we used variants of the hTelo sequence, where some guanines were substituted by 8-bromoguanine (BrG) derivatives, which preferentially adopt the syn orientation (42), allowing to achieve a stabilization of specific G4 conformations (18,43,44). The hybrid 2 structure is expected to be favored for a sequence containing two BrG substitutions at positions 9 and 14 (hTelo - BrG9,14) (44). Three E states were observed in the FRET histogram obtained for this sample (Figure 2B): a minor low E state representing an unfolded G4 and two high E states centered at E≈0.70 and E≈0.88, both corresponding to folded G4s. Taking into account the previous assignment of the E≈0.88 state to the chair conformation, the E≈0.70 state can be assigned to the hybrid 2 conformation. The hybrid 1 conformation is expected to be favored for the hTelo - BrG15 sequence (18). Three FRET peaks were also observed for this sample, with similar transfer efficiencies as for hTelo - BrG9,14 (Figure 2C). The highest FRET state is again attributed to the chair conformation and the dominant peak at E≈0.70 to the hybrid 1 conformation. The hybrid 1 and hybrid 2 conformations are structurally analogous (45) and would likely yield very similar FRET efficiencies, as observed here (Figure 2B and C). Thus, we conclude that the E≈0.73 state observed for the hTelo sequence is represented by a mixture of hybrid 1 and hybrid 2 conformations (Figure 1E). The hybrid 1 conformation is expected to be largely favored for the hTelo sequence (45). We therefore expect that the major long-lived subpopulation (E≈0.73) is likely to represent the hybrid 1 conformation, whereas the short-lived subpopulation can be attributed to the hybrid 2 conformation. Recent time-resolved NMR experiments suggest similar structural assignments for the G4s formed from an analogous telomeric sequence (29). It should be noted that the parallel G4 conformation appears at similar transfer efficiencies (E≈0.71) as the hybrid conformations (Supplementary Figure S6). Therefore, potentially it can also contribute to the E≈0.73 state of the hTelo sequence, however, the formation of a parallel conformation has been observed only in a crystal state or under dehydrating conditions and is unlikely to be observed under our experimental conditions (46,47).