Fluorescent strategy for detection of uracil-DNA glycosylase activity based on isothermal amplification triggered by ligase

Abstract

Uracil-DNA glycosylase (UDG) plays a key role in the base repair system, and detecting its enzymatic activity is crucial for early disease diagnosis. A rapid method for detecting UDG was developed, utilizing amplification initiated by a ligation reaction. A DNA probe modified with uracil was utilized to ligate two free DNA strands to form a newly generated DNA strand. This triggers a nicking enzyme-assisted amplification reaction, resulting in the production of single-stranded DNA (ssDNA). Then, the amplified ssDNA triggered the molecular beacons to emit fluorescence. However, the addition of UDG results in the removal of uracil from the DNA probe strand, leaving abasic site (AP site). After heat denaturation, this site was destroyed, preventing subsequent ligation or amplification reactions, resulting in the absence of fluorescence. The findings of our study indicate that the addition of UDG at concentrations exceeding 0.5 U/mL resulted in complete suppression of fluorescence intensity, reaching a value of 0. Conversely, in the absence of the UDG enzyme or upon the addition of other enzymes and proteins such as HAAG, EndoIV and BSA, the fluorescence intensity of the system remains unaffected, achieving 100 % intensity within 5–20 min. This study presents a rapid method for assessing UDG activity that could be valuable for early disease diagnosis in the future.