Three-Way Junction-Assisted Rolling Circle Amplification Integrated with trans-Cleavage of Cas12a for Sensitive and Reliable Detection of miRNA.

Abstract

MicroRNAs (miRNAs) serve a crucial role in numerous biological processes, such as acute pancreatitis development. Due to its low abundance and high similarity among homogeneous family members, sensitive and reliable detection of microRNA remains a formidable challenge. By combining the three-way junction-assisted rolling circle amplification (RCA) with the trans-cleavage of Cas12a, we propose a novel fluorescent technique for sensitive miRNA detection. In order to increase the amplification efficiency of RCA-based methods, catalytic hairpin amplification (CHA) is incorporated into the RCA process, playing the roles of specific target recognition and three-way junction formation. Consequently, the method demonstrated a six-orders-of-magnitude detection range and a LOD as low as 27 aM, making it a promising method for the early diagnosis of various diseases.