Proximity rolling circle amplification based generation of lighting-up aptamer for sensitive and label-free MicroRNA analysis

The dysregulated expression of microRNA (miRNA) has been strongly linked to pneumonia. However, ultrasensitive miRNA detection remains technically challenging due to their short sequences and low abundance in biological samples. Herein, we developed a catalytic hairpin assembly -triggered proximity rolling circle transcription system for synthesizing malachite green (MG)-binding RNA aptamers, enabling label-free and highly sensitive detection of miRNA-21. In this system, target miRNA-21 initiates hairpin probe structural rearrangement, promoting miRNA-21 recycling and activating dual rolling circle transcription reactions. The resulting long RNA transcripts undergo partial hybridization, yielding numerous functional MG RNA aptamers. Upon binding to MG dye, these aptamers generate a robust fluorescence signal, allowing ultrasensitive miRNA-21 detection at concentrations as low as 0.51 fM. The assay exhibits high specificity, discriminating miRNA-21 from single-base mismatched sequences, and shows promise for monitoring miRNA-21 expression in clinical samples. By combining the signal amplification of rolling circle transcription with the high-affinity recognition of MG aptamers, this method achieves a low background, superior signal-to-noise ratio, and exceptional sensitivity. Furthermore, the strategy can be readily adapted to detect other trace biomarkers by modifying the target-recognition sequence.