(A) label-free method to detect ribonuclease H activity based on fluorescent light-up aptamer

Abstract

Herein, we developed a label-free method to detect ribonuclease H (RNase H) by utilizing target-triggered amplification of fluorescent light-up aptamers. In principle, this system employs an RNA/DNA chimeric hairpin promoter sequence as a detection probe for RNase H activity assay. RNase H hydrolyzes RNA strand in the detection probe, releasing T7 promoter sequence to make complete double-stranded T7 promoter. Then, T7 RNA polymerase recognizes the structure and amplify a large number of broccoli aptamer, which is a type of fluorescent light-up aptamers, and they bind to the (Z)-4-(3,5-Difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI) fluorogenic dye and emit intense fluorescence signal. With this method, we successfully measured RNase H activity down to 0.000156 U/mL, with high selectivity against other nucleases and a DNA repair enzyme. Furthermore, we also demonstrated practical applicability of this strategy by successfully analyzing RNase H activity in a complex cellular extract environment.