Highly Effective Colorimetric and Visual Detection of Nucleic Acids Using an Asymmetrically Split Peroxidase DNAzyme

Abstract

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

“…Zhang and Winfree have shown that single-nucleotide mutations, insertions, and deletions in the catalyst strand can result in 10-to 300-fold reductions in its efficiency in catalyzing strand exchange [17]. These reductions in signal observed with single-nucleotide mismatches are comparable to other well-developed DNA sensors [26][27][28][31][32][33]. Consistent with these findings, we observed that when a single nucleotide was changed in the toehold region (Figure 3, inset), the mutant Trigger did not yield any peroxidase activity above background even at 150 nM concentration ( Figure 3, sample #10~12).…”

“…We also demonstrate that an entropy-driven catalytic DNA circuit can function as a generic signal amplification module that allows lower concentrations of input molecules to control the production of higher concentrations of deoxyribozymes, and thus exceed the limit of stoichiometric allosteric control. The system is qualitatively sensitive to mismatches in the input nucleic acid, suggesting that the combined entropy-driven strand exchange and allosteric deoxyribozyme circuit might facilitate the future development of enzyme-free, point-of-care diagnostic assay system [26][27][28].…”

Visual Detection ofrpoBMutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

“…In the detection system, the initial tool for SNP analysis is the molecular-beacon probe (19); however, the requirement for the instrument and the individually fluorescence-labeled probe is definitely costly. The peroxidase DNAzyme was selected due to its robustness, its sensitivity, and its relatively low cost (3,12,14,25,26,27,32). Here, we apply the 3:1 split DNAzyme to detect target DNA.…”

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

“…This method was further extended and, by combining with a personal glucose meter, a quantitative detection system of non-glucose targets was developed. 326,354 By entrapping gold nanoparticles in aptamer-based DNA hydrogels, proteins were also detected with a very high sensitivity. 355 When the target protein was recognized by the aptamer in the gel, the gel was broken down and the gold nanoparticles were released to the solutions.…”