Label-free and sensitive detection of uracil-DNA glycosylase using exponential real-time rolling circle amplification

• Published in: Analytical methods Vol. 10; no. 20; pp. 2405 - 2410
• Main Authors: Xu, Yuan, Cui, Yun-Xi, Zhao, Qiu-Ge, Tang, An-Na, Kong, De-Ming
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 01.01.2018
• Subjects: Amplification; analytical methods; biochemical pathways; Contamination; Deoxyribonucleic acid; DNA; DNA glycosylase; Endonuclease; Feasibility studies; Fluorescence; Nicking endonuclease; Oligonucleotides; prognosis; Real time; risk reduction; Strategy; Uracil; Uracil-DNA glycosidase; uracil-DNA glycosylase
• DOI: 10.1039/C8AY00742J
• ISSN: 1759-9660; 1759-9679

Sensitive evaluation of the uracil-DNA glycosylase (UDG) activity is greatly significant in both fundamental biochemical process studies and disease prognosis. In this study, a simple but sensitive UDG activity-sensing strategy was designed on the basis of UDG-triggered rolling circle amplification (RCA) reaction. In this strategy, two oligonucleotides were used. The hairpin-like structure of the oligonucleotide containing a uracil nucleotide is destroyed in the presence of UDG, and then can be employed to form the circular template of RCA and initiate the subsequent RCA reaction. The participation of a nicking endonuclease makes the RCA reaction proceed in an exponential amplification mode. The amplification product may fold into a G-quadruplex structure, which can be specifically combined with thioflavin T to generate a fluorescence signal without any extra label. This UDG activity-sensing strategy was demonstrated to work well in both end-point and real-time detection modes with high sensitivity and excellent specificity. As low as 5.5 × 10 −5 U mL −1 UDG could be detected. The advantages of simple operation, short RCA time and automatic measurement using commercial instruments make the real-time detection mode suitable for high-throughput detection with reduced risk of amplification product carryover contamination, and its application feasibility in real samples was demonstrated by UDG activity analysis of cell lysate.