Kinetic analysis of catalytic activity of G-quadruplex/hemin DNAzyme with flanking adenine nucleotides

G-quadruplex/hemin DNAzymes are promising nucleic acid catalysts due to their versatility and ease of use in biosensing applications. Their peroxidase-like catalytic activity can be enhanced through various strategies, including modifications to flanking nucleotides. In this study, the catalytic effects of flanking nucleotide modifications at the 3' and 5' ends of the DNAzyme were investigated. Additionally, the structural topology of the G-quadruplex/hemin DNAzymes was characterized using circular dichroism (CD) spectroscopy. Similar to the unmodified DNAzyme, the modified G-quadruplex with adenine (A) nucleotide at the 3'-terminal extension adopted a parallel topology in the presence of hemin. After optimizing the reaction conditions, the kinetic parameters of both original and 3' flanking A modified G-quadruplex/hemin DNAzymes were evaluated using the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in the presence of hydrogen peroxide (H₂O₂). The kinetic analysis revealed a significant enhancement in catalytic efficiency upon the addition of A nucleotides at the 3' end. Notably, the DNAzyme with a 3'-terminal AA modification exhibited approximately a ten-fold increase in catalytic efficiency compared to the unmodified form, as indicated by higher turnover numbers (k_cat) and lower H₂O₂ substrate affinity (K_m). This enhanced catalytic performance was further demonstrated by improved colorimetric signal detection of circulating tumor DNA (ctDNA), underscoring the potential of the modified DNAzymes for more sensitive detection in colorimetric biosensor applications.