Single-molecule transfer efficiency histograms of G4s formed from hTelo-m (A), hTelo - BrG9,14 (B), hTelo - BrG15 (C) and hTelo-I14 (D) sequences in 25 mM KCl. Measurements were performed on samples that were thermally annealed (A–C) or equilibrated for several hours (D). Histograms were fitted to one or a mix of three Gaussian functions (black lines). The color code used for identification of FRET peaks corresponding to different G4 conformations is the same as in Figure 1F. (E) Structure of one of the experimental DNA constructs with the hybrid 2 G4 structure (PDB ID: 2JSL) as obtained from molecular dynamics simulations. The positions of the donor and acceptor dyes were estimated using the available volume (AV) approach and are shown as green and red clouds, respectively. (F) Transfer efficiencies obtained from single-molecule FRET experiments (EsmFRET) and AV calculations based on the MD simulations (EAV). For each particular G4 conformation (see schematic structures) the EsmFRET values represent an average of mean transfer efficiencies obtained for hTelo and corresponding modified sequences. The EAV values are the average of transfer efficiencies for each G4 conformation estimated from four approaches of choosing input frames for the AV calculations (see Supplementary Figure S5 and ‘Materials and Methods’ section for details). The diagonal dashed line shows the identity line.