G-quadruplexes (G4) are four-stranded DNA structures formed by nucleic acid sequences that are rich in guanine.There is increasing evidence that G4 structures are formed in cells and may have detrimental effects on transcriptional regulation,DNA replication and repair,maintenance of telomere length and genome stability.It is therefore of fundamental and medical importance to unravel the molecular mechanism of G4 unfolding.We first employed magnetic tweezers to investigate the unfolding of single G4.The results are consistent with a model in which the G-triplex (G3) is an in-pathway intermediate in the folding of G4.