Highly sensitive detection of miRNA based on a quencher-free fluorescence strategy with dual-signal amplification

MicroRNAs (miRNAs) have emerged as crucial post-transcriptional regulators of gene expression and promising biomarkers for disease diagnosis and prognosis. Their aberrant expression levels are closely associated with the pathogenesis and progression of various human diseases. In this study, we developed a novel quencher-free dual-signal amplification strategy for highly sensitive miRNA detection. The approach involves the use of a target-specific padlock probe that undergoes miRNA-triggered circularization to initiate rolling circle amplification (RCA). The resulting RCA products then hybridize with 2-aminopurine-labeled hairpin probes, maintaining fluorescence quenching in the hybridized state. Upon introduction of exonuclease III, the enzyme-mediated cyclic cleavage enables duplex structures to release numerous 2-aminopurine molecules into the solution, generating a strong fluorescence signal through dual amplification (RCA and exonuclease III digestion). This innovative design eliminates the requirement for conventional quencher molecules while achieving high sensitivity, and the method has a detection limit of 63 aM for miRNA-21 and possesses single-base mismatch discrimination capability. The clinical applicability of the method was demonstrated through direct quantification of miRNA-21 in both human serum samples and tumor cell lysates without the need for total RNA extraction. These results validate the potential of the strategy for early cancer diagnosis.