Target-induced multipath-to-one-substrate approach for high-efficient bioanalysis of microRNA

Abstract

Considering the significant potential of microRNA (miRNA) as an efficient biomarker and great challenge of accurate analysis of lowly abundant miRNA, herein, we proposed a target-induced multipath-to-one-substrate strategy to monitor miRNA in vivo and in vitro accurately with high-efficient performances. In presence of target miRNA, it could directly generate the catalytic hairpin assembly (CHA) amplification cycle based on hybridizing with hairpin 1 (H1) and H2 respectively to structure the H1-H2 duplex, then the H1-H2 duplex could activate the cleavage ability of CRISPR/Cas12a to cleavage H1 which represent miRNA indirectly consume H1, which achieve co-consumption of the same substrate H1 by multiple pathways. And thus, the quenched fluorescent signal on H1 could be recovered due to the enlarger distance between fluorescent probe and quencher by the formation of H1-H2 duplex or cleavage of H1, all of which were related directly with target miRNA or indirectly with H1-H2 duplex activated cleavage ability of CRISPR/Cas12a, generating ultrahigh sensitive analytical ability and high-efficient analytical performances, such as more simple, fast, efficient and so on, especially a linear correlation from 100 pM to 100 nM with a detection limit of 78 pM, opening a new door to monitor expression level of biomolecules for early diagnosis and prognosis evaluation of various diseases.

Keywords: Co-substrate consumption; Nucleic acids; Real-time fluorescence assay; Signal amplification.