Exploring the catalytic mechanism of multivalent G-quadruplex/hemin DNAzymes by modulating the position and spatial orientation of connected G-quadruplexes.

G-quadruplex (G4)-hemin complexes are a convenient peroxidase mimicking DNAzyme for utilization in biosensing and analytical applications. Although dispersive G4/hemin DNAzymes have been extensively studied, a thorough investigation of the catalytic mechanism of multivalent G4/hemin (MultiG4) DNAzymes is warranted. To address this, dispersive G4/hemin DNAzymes with high-efficiency are connected by double- or multi-stranded DNA structures to build MultiG4 DNAzymes. The distance and environment of hemin binding sites are regulated by altering the position and spatial orientation of these connected G4s. Our data demonstrate that the catalytic activities of duplex-spaced MultiG4 DNAzymes are not affected by duplex length (within a reasonable range). However, vicinal MultiG4 DNAzymes that are immobilized at small spatial distances by Watson-Crick based DNA structures usually exhibit much lower catalytic activities than dispersive G4/hemin DNAzymes. Our results reveal that increasing the spatial flexibility of vicinal MultiG4 DNAzymes is imperative to achieving high catalytic efficiency. Significantly, we demonstrate that the catalytic activities of vicinal MultiG4 DNAzymes regulated by parallel duplexes are similar to that of dispersive G4/hemin DNAzymes, and that their activities are independent of the proximity effect. Thus, vicinal MultiG4 DNAzymes arranged in the same direction are more conducive to the maintenance of catalytic efficiency than those arranged in opposite directions. Our study provides a perspective for exploring multienzyme catalysis and should contribute to the design of nanozymes with high-efficiency catalytic activities.