Coexistence of G‐Quadruplex and i‐Motif Within a DNA Duplex is Tolerated by a PCBP2‐Assisted Replisome

Abstract G‐quadruplexes (G4s) and i‐motifs (iMs) are non‐canonical, four‐stranded DNA structures that are formed in guanine‐ and cytosine‐rich sequences, respectively. These structural motifs play crucial regulatory roles in diverse genomic processes and have emerged as promising therapeutic targets. Although individual G4s and iMs have been intensively characterized, whether they could form simultaneously on complementary DNA strands has remained unclear. Using single‐molecule analysis, we demonstrate that under physiologically relevant conditions, G4 and iM structures can indeed coexist within a native DNA fragment comprising 24 guanine and cytosine bases. Strikingly, due to the intrinsic sequence complementation, the stabilities of the two motifs are mutually affected, giving rise to a tightly coupled unfolding pathway. Quantitative measurements reveal that iM unfolding attenuates the mechanical stability of the facing G4 by approximately 13.7 kcal mol −1 . Furthermore, we identify an oncogenic splicing factor, PCBP2, as an iM‐interacting protein. Through destabilizing iM and consequently the concomitant G4, PCBP2 enables a replisome to bypass these otherwise persistent barriers. Our findings provide fresh insights into G4–iM interplay and establish PCBP2 as a key regulator of structured DNA during genome maintenance.