G-Quadruplexto Duplex Conversion: The Roleof Guanine Quartet versus Loop

Abstract The structural interconversion between the G-quadruplex (G4) and duplex DNA is central to many biological processes, yet the factors governing this transition remain incompletely understood. Here, we systematically examine the roles of guanine (G)-tract length and loop architecture in modulating the kinetics of the G4-to-duplex transition using circular dichroism (CD), NMR spectroscopy, gel electrophoresis and time-dependent FRET measurements. Our results show that increasing the number of guanines in the G-tract significantly enhances the G4 stability and reduces the propensity for duplex formation. Sequences with shorter G-tracts undergo rapid conversion to duplex upon the addition of complementary strands, whereas those with longer G-tracts exhibit slower kinetics or remain predominantly in the G4 state. Binding studies reveal that the affinity of the complementary strand decreases markedly with an increasing G-tract length, establishing an inverse correlation between G4 stability and duplex formation. Kinetic analyses further indicate the presence of multiple G4 conformational populations that interconvert at distinct rates, reflecting the structural heterogeneity in the G4 ensemble. In contrast, variations in loop length exert a relatively modest and nonmonotonic effect on binding affinity but significantly influence the kinetics of the transition. Increasing the loop length reduces structural inhomogeneity and enhances the contribution of the fast-converting population. Overall, these findings demonstrate that the G-tract length is the dominant factor controlling G4 stability and duplex conversion, whereas the loop length and composition fine-tune the kinetic pathways by modulating the underlying conformational landscape. This study provides molecular-level insight into the role of the G-tract and loop nucleobases of the G4 in the structural transition from the G4 to the duplex.