One-step G-quadruplex-based fluorescence resonance energy transfer sensing method for ratiometric detection of uracil-DNA glycosylase activity

Abstract

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.