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

Abstract

Uracil‐DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme‐powered, label‐free DNA walker was devised for UDG activity detection. Initially, a label‐free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G‐quadruplex sequence, enabling the generation of a label‐free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme‐powered walking process and generating significant dissociative G‐quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label‐free fluorescence response was achieved. The proposed DNA walker presented a direct and label‐free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single‐cell level was accomplished. Consequently, the developed DNA walker serves as a label‐free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.