Catalytic single-molecule Förster resonance energy transfer biosensor for uracil-DNA glycosylase detection and cellular imaging

Uracil-DNA glycosylase (UDG) is essential to the maintenance of genomic integrity due to its critical role in base excision repair pathway. However, existing UDG assays suffer from laborious procedures, poor specificity, and limited sensitivity. In this research, we construct a catalytic single-molecule Föster resonance energy transfer (FRET) biosensor for in vitro and in vivo biosensing of UDG activity. Target UDG can remove uracil base from the detection probe and cause the cleavage of detection probe by apurinic/apyrimidinic endonuclease (APE1), which exposes its toehold domain and initiates catalytic assembly of two fluorescently labeled hairpin probes via toehold-meditated strand displacement reaction (SDA) to generate abundant DNA duplexes with amplified FRET signal. In this assay, target UDG signal is amplified via enzyme-free catalytic reaction and the whole reaction may be completed in one step, which greatly simplifies the assay procedure, reduces the assay time, and facilitates the cellular imaging. This biosensor enables specific and sensitive measurement of UDG down to 0.00029 U/mL, and it is suitable for analyzing kinetic parameters, screening inhibitors, and even imaging endogenous UDG in live cells. Importantly, this biosensor can visually quantify various DNA repair enzymes by rationally altering DNA substrates.