Triple DNAzyme cleavage mediated signal cascade for sensitive and reliable Kawasaki disease related microRNA analysis.

MicroRNAs (miRNAs) serve as promising biomarkers for disease diagnosis, therapeutic monitoring, and post-treatment surveillance. However, their accurate quantification remains challenging due to low abundance and sample-derived interference. To address this, we developed an enzyme-free DNAzyme cascade system for highly sensitive miRNA detection. This approach employs programmable DNAzyme hairpin probes (S1, S2, and S3), where the S1 probe features exposed recognition subunits for target-specific miRNA binding. This recognition initiates two steps: the split DNAzyme-mediated middle circuit and the subsequent substrate cleavage catalyzed by DNAzyme to induce signal generation (downstream DNAzyme circuit). The absence of enzymes provides the method with a negligible background signal. The numerous signal cycles facilitated significant signal amplification, resulting in a femtomolar detection limit and enhanced selectivity for several homologous miRNAs. This robust triple DNAzyme cascaded system provides enhanced and reliable approaches for understanding miRNA activity in diverse biological events.