A label-free fluorescence method for the detection of uracil DNA glycosylase activity based on G-quadruplex formation

Abstract

Here, we have developed a novel fluorescence strategy for sensitive detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation by using a label-free DNA hairpin probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the hairpin DNA, resulting in the conformational transition of the hairpin structure. Then, the probe DNA can be recognized quickly by the thioflavin T (ThT) dye resulting in an increase in fluorescence. This strategy could detect UDG activity as low as 0.01 U mL⁻¹. In addition, the strategy was also applied for the detection of UDG activity in HeLa cell lysate. It is simple and of low cost without the requirement of labeling with a fluorophore–quencher pair. Furthermore, UDG activity inhibition by uracil glycosylase inhibitor (UGI) is shown, demonstrating the potential for the discovery and characterization of UDG-targeted drug candidates in pharmaceutical development.