Target-triggered activation of rolling circle amplification for label-free and sensitive fluorescent uracil-DNA glycosylase activity detection and inhibition.

The expression variations of uracil-DNA glycosylase (UDG) can be used as effective biomarkers for the evaluation of gene regulation and related diseases. Here, by using a new target-triggered activation of rolling circle amplification (RCA) signal enhancement strategy, we have established a sensitive and label-free fluorescent approach for UDG activity detection and inhibition. The target UDG specifically recognizes and excises the uracil bases in a three-strand containing DNA complex to liberate one of the strands. Subsequent ligation of the excised DNA complex converts it into a suitable primer/circular template structure for the initiation of RCA for the generation of long DNA sequences with many repeated G-quadruplexes. Protoporphyrin IX further binds these G-quadruplexes to show substantially enhanced fluorescence to achieve sensitive detecting the activity of UDG with the detection limit as low as 0.00014 U mL-1. Besides, this assay approach has a high specificity toward UDG and can also be utilized to evaluate its inhibition by the uracil glycosylase inhibitor, highlighting the promising applications for convenient and sensitive UDG activity detection and inhibition for disease diagnosis and drug screening.