Colorimetric Assay for Uracil DNA Glycosylase Activity Based on Toehold‐Mediated Strand Displacement Circuit

Abstract

Herein, a novel enzyme‐free and label‐free strategy for colorimetric assay of uracil DNA glycosylase (UDG) activity, which relies on a target‐activated toehold‐mediated strand displacement (TMSD) circuit is described. The strategy employs a detection duplex probe composed of a uracil‐containing strand (US) and a catalyst strand (CS). UDG present in a sample will cleave uracil bases within US and destabilize the detection duplex probe, which then leads to the dissociation of the detection duplex, releasing CS. The free CS promotes the TMSD reaction, consequently liberating a G‐quadruplex DNAzyme strand (GS) which is initially caged by a blocker strand (BS). Notably, a fuel strand (FS) is supplemented to recycle the CS to promote another cycle of TMSD reaction. As a consequence, a large number of GSs are activated by UDG activity and a distinct colorimetric signal is produced through the oxidation of ABTS promoted by the peroxidase mimicking activity of the liberated GSs. Based on this design principle, UDG activity down to 0.006 U mL−1 with excellent selectivity is successfully determined. The practical applicability of this assay is also demonstrated by reliably determining UDG activities in human serum.

Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity

“… three-step protocol, approx. 75 min (measurement not included) plate reader, smartphone or spectrophotometer colorimetric [ 31 ] Detection of colorimetric signal produced by peroxidase activity of quadruplex and hemin after dissociation of non-labelled double-stranded probe and Toehold-mediated strand displacement circuit. 6 mU ml −1 n.a.…”

“…TIRF)luminescence [32]Detection of luminescence after formation of G-quadruplex from non-labelled double-stranded probe and binding of small organic molecule (DID-VP) to ON.5 mU ml −1 n.a.n.a.two-step protocol, >30 min (measurement not included)fluorescence plate reader or spectrophotometercolorimetric [33]Detection of colorimetric signal after formation of G-quadruplex from non-labelled double-stranded probe and signal generation by peroxidase activity of quadruplex and hemin.8 mU ml −1 n.a.n.a.three-step protocol, approx. 75 min (measurement not included)plate reader, smartphone or spectrophotometercolorimetric [31]Detection of colorimetric signal produced by peroxidase activity of quadruplex and hemin after dissociation of non-labelled double-stranded probe and Toehold-mediated strand displacement circuit.6 mU ml −1 n.a.n.a.four-step protocol, approx. 75 min (measurement not included)plate reader or spectrophotometer…”

“…The measurement of UNG activity can be realized by various strategies [25][26][27][28][29][30][31][32][33][34][35][36]. They include approaches based on the combination of FAM-labelled hairpin DNA probe, exonuclease treatment and graphene-induced quenching [30], employing G-quadruplex formation [32,33] or using singlemolecule detection [34].…”

Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein

“…TMSD’s fluorescence-based detection relies on base pairing between the sticky nucleic acids and the toehold region, allowing the theoretical detection of different targets in a single tube using G strands labeled with different fluorophores, and the extent of multiplexing is determined by the availability of suitable fluorophores for labeling probes. Here, the use of TMSD has offered a typical approach for easy and low-cost molecular logic systems and showed an orthogonal capacity. − Therefore, the CRISPR-TMSD assay appears to be promising for multiplexing detection. In contrast, previous systems, such as SHERLOCKv2, using the trans -cleavage activities of Cas, require a Cas protein for each target, limiting the number of detection targets due to the scarcity of known Cas proteins and the increased complexity of the system. , …”

“…In a typical TMSD, the binding of the invader strand to the duplex’s toehold initiates the reaction, displacing the incumbent strand via branch migration. TMSD has been widely applied in DNA biosensing due to its rapid kinetics and excellent sequence specificity. − Furthermore, to improve the sensitivity of TMSD, several cascade reactions triggered by the target nucleic acid have been developed. , For example, the nonenzymatic isothermal strand displacement and amplification (NISDA) assay and the sensitive splint-based one-pot isothermal RNA detection (SENSR) assay were developed for high-sensitivity detection of SARS-CoV-2. , …”