Rational redesign of high-activity G-quadruplex DNAzyme through flanking and looping of nucleobases.

G-quadruplex (G4) DNAzymes are guanine-rich oligonucleotides with intrinsic peroxidase-mimicking activity upon complexation with hemin, offering a promising alternative to protein-based enzymes in biosensing. However, their relatively low catalytic efficiency limits practical applications. Here, we present a structure-guided redesign of the high-activity aptamer [B7]-3-0 by incorporating strategic flanking and looping nucleobase modifications. Introduction of adenine and thymine-cytosine elements at the 3' end led to up to 4-fold enhancements in reaction extent and a 3-fold increase in initial velocity under moderate hydrogen peroxide conditions (0.425 mM). Remarkably, the modified B730 variants retained activity at elevated H₂O₂ concentrations (4.25 mM), achieving up to 8-fold catalytic enhancement and outperforming high-activity DNAzymes including AS1411 and CatG4. These redesigned DNAzymes demonstrated improved peroxidase activity and resistance to oxidative inactivation, addressing a major limitation of both natural and artificial peroxidases. Our findings establish flanking and loop engineering as a cost-effective and broadly applicable strategy for optimizing G4 DNAzymes and underscore their potential in the development of next-generation biosensors.