Label-free colorimetric assay for base excision repair enzyme activity based on nicking enzyme assisted signal amplification

摘要

Specific and sensitive detection of base excision repair enzyme activity is essential to many fundamental biochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized uracil DNA glycosylase (UDG) activity assay based on nicking enzyme assisted signal amplification. In this method two hairpin probes were employed for the colorimetric detection, namely hairpin probe 1 (HP 1) carrying two uracil residues in the stem, and hairpin probe 2 (HP 2) containing a G-riched DNAzyme segment, and the recognition sequence as well as the cleavage site for the nicking enzyme. In the presence of UDG, the uracil bases in the stem of HP 1 can be specifically recognized and hydrolyzed by UDG, which leads to the destabilization of its stem containing abasic sites (AP sites), and then results in the opening of HP 1 to form a single strand. The opened HP 1 hybridizes with HP 2 to form a DNA duplex, which initiates the specific cleavage of HP 2 by the nicking enzyme, leading to the release of G-riched DNAzyme segments. As a result, HP 1 is released and able to hybridize with another HP 2 to induce the continuous cleavage of HP 2, generating enormous amount of G-riched DNAzyme segments. Finally, the G-riched DNAzyme segments bind hemin to form a catalytically active G-quadruplex-hemin DNAzyme which can catalyze the H_2O_2-mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS~(2-)) to the colored ABTS~-, providing a visible signal for UDG activity detection. This assay exhibits several advantages such as simplicity, low-cost, high selectivity and desirable sensitivity, which shows great potential of providing a promising platform for convenient and visualized analysis of UDG or other biomolecules.