Inactivation Kinetics of G‐Quadruplex/Hemin Complex and Optimization for More Reliable Catalysis

Abstract

Reliable catalysis is critical for the synthesis of various chemicals, molecular sensing and biomedicine. G-quadruplex/Hemin (GQH) complex, a peroxidase-mimicking DNAzyme, has been widely used in various publications. However, a concern exists about the unstable kinetics of GQH-catalyzed peroxidation. This work investigates several factors that result in the inactivation of GQH and the signal degradation during long reaction periods, including pH, buffer component, the selection of substrate and the oxidation damage of cofactor. Using colori-metric and fluorescent assays, GQH was found to be highly unstable under basic conditions with 50 % of GQH activity lost within 2 minutes at high H 2 O 2 concentrations. Appropriate conditions and substrates are suggested for accurately characterizing GQH-catalyzed reactions, as well as optimization to improve the catalytic reliability, such as the use of polyhistidine and cascade reactions. These results could be useful for GQHrelated applications.

Ferric hemebin aqueous micellar and vesicular systems: state-of-the-art and challenges

“…from amounts of products obtained at reaction equilibrium. This important issue has been discussed in some of the many investigations of G-quadruplex/hemin complexes (Yang et al ., 2011; Monte Carlo and Fu, 2022) in which it was concluded that high initial reaction rates may not correlate with high reaction yields due to apparent inactivation of the G-quadruplex/hemin complexes.…”

Optimization and Enhancement of the Peroxidase-like Activity of Hemin in Aqueous Solutions of Sodium Dodecylsulfate

“…Concerning the leveling-off of the amount of formed CTC after 5 min of reaction (Figure A), three possible explanations are (i) the amount of H 2 O 2 added at the beginning of the reaction was limiting; (ii) H 2 O 2 caused hemin inactivation (degradation), as was reported to be the case for G-quadruplex/hemin systems , ; and (iii) the CTC interacted with hemin thereby preventing the reaction from proceeding further (product inhibition). Possibility (i) could be excluded by one of the experiments reported in Figure S3 in the Supporting Information: For the reaction run in 100 mM HEPES (pH = 7.2), addition of a new portion of H 2 O 2 after 5 min of reaction did not increase the CTC yield, excluding H 2 O 2 as a limiting factor.…”

“…The use of apoHRP-mimicking structures different from apoHRP in combination with hemin or hemin derivatives to achieve peroxidase-like activity in vitro is a challenge that various research groups have addressed in the past and are still addressing . Apart from using (i) proteinaceous scaffolds with one or more hydrophobic binding sites, ,− (ii) sophisticated G-quadruplex RNA and DNA structures for obtaining so-called “RNAzymes” or “DNAzymes,” − or (iii) hemin-metal–organic frameworks (MOFs) systems, , one idea is (iv) to use aqueous micelles ,,− or vesicles composed of chemically simple amphiphiles to host hemin in catalytically active state . This type of research on hemin-based, peroxidase-mimicking systems is of interest for potential biotechnological applications as cheap catalysts, for example, for oxidative oligo- or polymerization reactions. − Furthermore, assemblies of chemically simple amphiphiles and iron porphyrins might have played a role as primitive catalysts in prebiotic protocellular structures, before the first living cells emerged from nonliving forms of matter, at the origin of life …”

CRISPR/Cas14 and G-Quadruplex DNAzyme-Driven Biosensor for Paper-Based Colorimetric Detection of African Swine Fever Virus

“…Our results also showed that G4 showed better binding performance to hemin under 25 °C than that of 37 °C (Figure 3f). 38,39 Thus, the optimal incubation temperature was set at 25 °C for this cascade detection. Sensitivity of the CRISPR/Cas14a-G4 Biosensor.…”