Single G-quadruplex-based fluorescence method for the uracil-DNA glycosylase inhibitor screening

Abstract

Uracil-DNA glycosylase (UDG) plays a pivotal role in the base repair system. Through bioinformatics, we found that the expression of the UDG enzyme in many cancer cells is increased, and its high expression is not conducive to the prognosis of lung cancer patients. The development of analytical techniques for the quantification of UDG activity and the identification of UDG inhibitors is of paramount importance. We found that when the T base in the G4 loop region mutated to uracil, the G4 structure was not disrupted and still retained the characteristics of a G4 structure (emitting strong fluorescence after binding with ThT (Thioflavin T). Inspired by this phenomenon, we developed a detection method for UDG and its inhibitors utilizing a single DNA strand engineered to form a G-quadruplex structure, containing uracil residues within the loop region, designated as G4-dU. The inclusion of uracil-DNA glycosylase (UDG) in the assay environment induces the removal of uracil, resulting in the formation of apurinic sites (AP) within the G4-dU sequence. Subsequent thermal denaturation leads to strand cleavage at AP sites, precluding the reformation of the G-quadruplex configuration and abrogating fluorescence emission. The detection process in this study can be completed in only 30 min to 1 h, offers a straightforward, expedient, and efficacious modality for assessing UDG activity and UDG inhibitor potency, thereby facilitating the discovery of novel therapeutic agents for cancer treatment.

Keywords

• Uracil-DNA glycosylase
• G-quadruplex
• UDG inhibitors
• Detection