How Proximal Nucleobases Regulate the Catalytic Activity of G-Quadruplex/Hemin DNAzymes

G-quadruplexes (G4s) are versatile catalytic DNAs when combined with hemin. Despite the repertoire of catalytically competent G4/hemin complexes studied so far, little is known about the detailed catalytic mechanism of these biocatalysts. Herein, we have carried out an in-depth analysis of the hemin binding site within the G4/hemin catalysts, providing the porphyrinic cofactor with a controlled nucleotidic environment. We intensively assessed the position-dependent catalytic enhancement in model reactions and found that proximal nucleobases enhance the catalytic ability of the G4/hemin complexes. Our results allow for revisiting the mechanism of the G4/hemin-based catalysis, especially gaining insights into the rate-limiting step, demonstrating how both the G4 core and the proximal nucleotides dA and/or dC concomitantly activate the Compound 0 → 0∗ prototropic cleavage of H₂O₂ to foster Compound 1 formation. These results provide mechanistic clues as to how the properties of G4-based catalysts can be improved to ultimately make them competitive with proteinaceous enzymes.