A ligation DNAzyme-driven catalytic hairpin assembly strategy for miRNA detection

MicroRNAs (miRNAs) are emerging biomarkers for clinical diagnosis, yet their short length, low abundance, and sequence similarity pose substantial challenges for reliable detection. Here, we propose a ligation DNAzyme with catalytic hairpin assembly (LiD-CHA) as an alternative amplification strategy for sensitive and specific miRNA analysis. Unlike conventional CHA-DNAzyme systems that rely on cleavage-type DNAzymes and often suffer from background leakage, LiD-CHA employs a ligation DNAzyme to generate new trigger sequences, thereby minimizing nonspecific interference and enhancing detection accuracy. This design achieves enhanced signal amplification and a lower detection limit while retaining the inherent advantages of enzyme-free isothermal amplification. As a proof of concept, LiD-CHA was applied to detect microRNA-21 (miRNA-21) in blood samples, demonstrating the capability to differentiate cancer patients from healthy individuals. The results demonstrated performance comparable to RT-qPCR, underscoring the potential of LiD-CHA as a robust and accurate platform for clinical miRNA diagnostics.