Characterization of G‐Quadruplex/Hemin Peroxidase: Substrate Specificity and Inactivation Kinetics

Abstract

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.

Fabrication of Defined Polydopamine Nanostructures by DNA Origami‐Templated Polymerization

“…In the next step, DNAzyme on DNA nanotile was activated by incorporating its cofactor, hemin, resulting in no morphological differences by AFM (Figure S3c), but a characteristic shift of the hemin Soret band was detected in the UV/Vis spectra (Figure S4) that corresponds to existing literature 19. The catalytic activity of DNAzyme domain on DNA nanotile was evaluated by tracking the oxidation of 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid (ABTS) in the presence of H 2 O 2.…”

Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

“…Dz-11 showed the instant disappearance of E band (∼500 nm) and D band (∼630 nm) concomitant with the obvious increase in absorbance over 550–620 and 650–700 nm in 0.5 min after the reaction started (Figure 3C), indicating the generation of initial intermediate of G4–hemin complex (41). Sequentially, the overall decay over A450-700 nm occurred, evidencing the hemin degradation caused by successive reaction with H 2 O 2 (42). In contrast, in the case of Dz-00, the gradual diminishing of E and D bands as well as the slight increase of A 550-620 nm and A 650-700 nm were observed in the initial period of the reaction (0–2.5 min), implying a much slower formation of the initial intermediate of Dz-00 than Dz-11 (Figure 3C and D).…”

Biochemical and biophysical understanding of metal ion selectivity of DNAzymes

“…They also proposed a possible catalytic mechanism and inactivation pathway for G-quadruplex/hemin-dependent DNAzymes (shown in Figure 5d). 98 …”

“…Adapted with permission from ref 96 ; ( d ) A proposed mechanism of reaction and inactivation of G-quadruplex/hemin DNAzyme. Figure adapted with permission from ref 98 .…”