Enzyme-Powered, Label-Free DNA Walker for Uracil-Dna Glycosylase Detection at Single-Cell Level

Uracil -DNA glycosylase (UDG) is responsible for the excision of damaged uracil bases, maintaining genetic stability and integrity. Here, an enzyme-powered, label-free DNA walker was developed for UDG activity assay. First, a label-free DNA track for the walking process was designed with a gold nanoparticle (AuNP), multiple hairpin structures served as signal output components, and several swing arms acted as target recognition elements. The hairpin structure was specially designed with G-quadruplex sequence to achieve a label-free fluorescence signal. The swing arm was silenced without the UDG. While it could be activated through the addition of UDG, along with the initiation of the enzyme-powered walking process, generating substantial and dissociative G-quadruplex sequences. By inserting the pronounced structural selective fluorescent dye (NMM), improved and label-free fluorescence response was achieved. The proposed DNA walker provided a simple and label-free method for UDG detection and indicated an excellent sensitivity with a detection limit of 0.00004 U/mL. Moreover, taking uracil glycosylase inhibitor (UGI) as inhibitor model, inhibitor assay was obtained with acceptable accuracy. Furthermore, cellular UDG analysis was achieved at single-cell level, and UDG activity in Hela cell extracts was quantified. Therefore, the proposed DNA walker provided a label-free, selective, and sensitive tool for UDG activity assay, demonstrating a great potential for further application in early diagnosis of disease, inhibitor screening, and biomedical research.