Twice Target-Recognition Mediated Exonuclease iii (Exo-iii)-Propelled Cascade Signal Recycling MicroRNA Detection System with Improved Accuracy

Simple yet specific miRNA detection remains an enormous challenge due to its low abundance in samples and the high similarity among homologous miRNAs. Here, we propose a novel fluorescent approach for miRNA detection with greatly elevated accuracy by utilizing exonuclease-iii (Exo-iii) assisted twice target recognition. The proposed method involves a "Sensing probe" engineered with two loop sections to facilitate dual target miRNA recognition. The collaboration between Exo-iii and miRNA initiates target recycling for signal amplification, resulting in the formation of complete DNAzyme. The intact DNAzyme connects with the "Signal probe" and creates a nicking site within its loop region. The fluorescence signal of the "Signal probe" reemerges, correlating with the quantity of miRNA in the sensing system. The suggested technique demonstrates great selectivity for target miRNA and can readily differentiate sequences with a one-base mismatch, based on dual-target identification. Furthermore, the Exo-iii-assisted signal recycling imparts the approach with great sensitivity and a low detection limit of 548 aM. This method has the potential to be a robust alternative for the detection of miRNAs in real samples due to its high accuracy, simplicity, and resistance to potential fluorescence interferences.