a, Schematic representation of a G-tetrad (left) and a G4 structure (right). b, Chemical structures of the selective G4-fluorogenic ligand SiR-PyPDS (1) and its inactive isomer, SiR-iPyPDS (2). c, Schematic of the methodology used for visualizing individual G4s. Pre-folded G4 MYC is attached to a coverslip via a biotin–neutravidin linker. The fluorescent G4 probe SiR-PyPDS (1) binds to G4 MYC, which can be visualized using single-molecule fluorescence imaging. d, SiR-PyPDS will not bind single-stranded mutated-MYC that cannot form a G4. e, The inactive isomer SiR-iPyPDS (2), with its 10-fold weaker binding affinity, is less likely to bind G4 MYC. f, Quantification of SiR-PyPDS (1) binding to the G4 MYC, SiR-PyPDS (1) binding to the mutated-MYC (Mut), SiR-iPyPDS (2) binding to the G4 MYC and SiR-PyPDS (1) binding to the G4 MYC in the presence of 10 μM unlabelled PhenDC3 competitor. Error bars indicate mean ± s.d. ***P < 1 × 10−5, unpaired two-sided t-test, n = 12 measurements from three independent replicates. g, Representative images (500-ms exposure) of individual SiR-PyPDS (1) molecules (250 pM) binding to a surface coated with pre-folded MYC G4 oligonucleotide. Individual fluorescent puncta indicate binding of single SiR-PyPDS (1) molecules. h, SiR-PyPDS (1) (250 pM) binding to mutated-MYC. i, SiR-iPyPDS (2) (250 pM) binding to pre-folded MYC. Experiments in g–i were repeated three times independently with similar results. j, Interactions of G4 ligands and G4s can alter the equilibrium between unfolded and folded G4s. Error bars indicate mean ± s.d. n = 12 measurements, from three independent replicates. Changes in the Förster Resonance Energy Transfer (FRET) ratio can be observed at micromolar PDS concentrations for c-KIT1 and h-TELO and larger concentrations for MYC, indicative of G4 induction, which does not occur at lower concentrations.