Multiplex miRNA quantification based on response of difunctional molecular beacon to hairpin probe-triggered...

miRNA, the non-coding RNA comprised of about 22 nucleotides, serves as the biomarker in various biomedical scenarios based on different expression level. The more miRNAs are characterized, the more accurate result will be obtained, highlighting the necessary of multiplex miRNA quantification. Although PCR-based methods have been widely applied, the limited specificity and sensitivity restrict their application in liquid biopsy, prenatal diagnosis and forensic identification due to the trace of target and degraded specimen. Despite the higher sensitivity and specificity of loop-mediated isothermal amplification (LAMP), the short fragment renders miRNA detection impossible, let alone multiplex quantification. In this study, a novel strategy for multiplex miRNA quantification is developed based on the response of difunctional molecular beacon to hairpin probe-triggered isothermal amplification. Two hairpin probes are designed to hybridize with target miRNA and fused by ligase, forming a dumbbell structure as the trigger to initiate LAMP. The difunctional molecular beacon, featured with 3'-overhang, serves as loop primer by annealing to the dumbbell structure and participates in amplification. The quencher-fluorophore pair located on molecular beacon is separated through strand displacement, generating fluorescence to monitor amplification of each target for multiplex miRNA quantification. Hairpin probe addresses the incompatibility between template length requirement of LAMP and short fragment of miRNA, even extended cDNA from reverse transcription. Besides, specificity is improved by hairpin probe fusion with single nucleotide distinguishability, which is also not possessed in reverse transcription. The paradigm contains universal hairpin probe and molecular beacon, providing a general platform for multiplex miRNA analysis.