Colorimetric detection of uracil DNA glycosylase activity based on target-activated toehold-mediated strand displacement circuit

Abstract

Herein, we describe 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. The strategy employs a detection probe composed of an uracil-containing strand and a catalyst strand (CS) designed to trigger the TMSD circuit. Thus, in the presence of UDG which cleaves uracil bases and destabilizes the detection probe, CS is released to promote the TMSD reaction. This leads to the liberation of a G-quadruplex DNAzyme strand (GS) with the peroxidase mimicking activity, which is initially caged by a blocker strand. Notably, a fuel strand is supplemented to promote another cycle of TMSD reaction by recycling the CS, which activates a large number of GSs. As a consequence, a distinct colorimetric signal is generated from the oxidation of ABTS by GSs. With this strategy, we selectively determined the UDG activity down to 0.006 U/ml, and also identified its presence with the naked eye. Finally, the practical applicability of this assay was successfully demonstrated by reliably determining UDG activities in human serum.