Insights into how nucleotide supplements enhance the peroxidase-mimicking DNAzyme activity of the G-quadruplex/hemin system

Abstract

Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).

Incorporating ATP into biomimetic catalysts for realizing exceptional enzymatic performance over a broad temperature range

“…Altogether, the enhanced activity may be related to ATP as an H 2 O 2 activator and electron transfer energizer during the catalytic process. 35 Although the detailed mechanism for the boosting effect of ATP remains unclear, all of the results described above confirmed our hypothesis and indicated that ATP plays a very positive role in boosting catalytic processes over a wide range of temperatures.…”

“…Previous reports indicated that ATP may mimic the distal histidine residue of the peroxidase enzyme. 35 In addition, our results demonstrated that the non-enzymatic hydrolysis of ATP could take place under our experimental conditions (Supplementary Figure S13). Altogether, the enhanced activity may be related to ATP as an H 2 O 2 activator and electron transfer energizer during the catalytic process.…”

“…Thus, the use of enzyme mimics has been limited by their low catalytic activity at high temperatures. However, it is well known that ATP is not only considered to be the ubiquitous energy currency of life, 35 but is also well known to form charge transfer complexes with aromatic molecules and to participate in free radical redox chemistry. [36][37][38][39] For instance, the positive effects of ATP on ribonucleotide reductase-mediated catalytic reactions have been reported.…”

“…Inspired by the recent reports from Monchaud and colleagues 35 and Kong et al, 39 we assumed that ATP could also be used as an effective modulator to inhibit disproportionation and stabilize cationic free radical ABTS Áþ at high temperature. 35,39 To evaluate the feasibility of our presumption, we explored the effects of ATP concentration on the thermal stability of the reaction product in the presence of 60 mM H 2 O 2 . As shown in Figures 3c and d, the disproportionation of ABTS Á þ is gradually inhibited with increasing ATP concentration.…”

Improved method for chemiluminescent determination of peroxidase-mimicking DNAzyme activity

“…On the other hand, complexes of hemin and its G-quadruplex aptamers named peroxidase-mimicking DNAzymes (PMDNAzymes) 1 possess significantly higher catalytic activity [11][12][13][14]. This feature allowed them to be used in the construction of various analytical assays and sensors [15][16][17][18][19].…”