A novel fluorescence amplification strategy combining cascade primer exchange reaction with CRISPR/Cas12a system for ultrasensitive detection of RNase H activity

Abstract

Ribonuclease H (RNase H), which plays a vital role in various cellular processes, is to be closely related to the emergence of many diseases. As an essential therapeutic target, it shows great prospects in the development of associated drugs. Herein, a DNA-RNA chimeric hairpin (DR HP) was designed to introduce a new signal amplification strategy based on cascade primer exchange reaction (cPER) and CRISPR/Cas12a system for sensitive and specific analysis of RNase H activity. In the presence of RNase H, the RNA fragment of DR HP was specifically degraded and the blocked primer DNA was released. The process of enzymatic hydrolysis of substrate hairpin and cyclic signal amplification was completed in a one-step method under isothermal conditions, enriching many activator strands to initiate trans-cleavage of CRISPR/Cas system, thereby restoring the fluorescence signal. Under optimized conditions, the developed strategy exhibited a good linear relationship ranging from 0.005 to 0.1U/mL and offered a detection limit of 0.00061U/mL. Moreover, this method was used for RNase H activity assay in complicated human serum and real cell lysates with good stability and repeatability, and was also demonstrated to apply for RNase H inhibitors screening and inhibitory capability assessment. Therefore, the proposed system is a promising platform not only for determination of RNase H activity, but open up new thoughts for the biological enzyme research and inhibitor screening.

Keywords: CRISPR/Cas12a; Cascade primer exchange reaction; Fluorescence amplification; Ribonuclease H (RNase H); Ultrasensitive.