An intramolecular DNAzyme-based amplification for miRNA analysis with improving reaction kinetics and high sensitivity

Sensitive, specific and rapid methods for detecting microRNAs (miRNAs) play critical roles in disease diagnosis and therapy. Enzyme-free amplification techniques based on DNAzyme assembly have recently been developed for the highly specific miRNA analysis. However, traditional DNAzyme-based assembly (free DNAzyme) am-plifiers is mainly dependent on the target-induced split DNAzyme fragments to assemble into activated DNAzyme structures, which have made a compromise between the sensitivity and specificity due to the random diffusion of dissociative probes in a bulk solution with poor kinetics. Herein, based on a rationally designed DNA probe, we developed an intramolecular DNAzyme assembly (intra-DNAzyme) method to overcome these challenges. The miR-373 is used as model analyte for our current proof-of-concept experiments. Compared with the free-DNAzyme method, our method showed significantly improved analytical performance in terms of dynamic range, assay sensitivity and speed. This method can detect miR-373 specifically with a detection limit as low as 4.3 fM, which is about 83.7 times lower than the previous free-DNAzyme method. This intra-DNAzyme strategy would be of great value in both basic research and clinical diagnosis.

Automated summary

Sensitive, specific, and rapid methods for detecting microRNAs (miRNAs) are crucial for disease diagnosis and therapy. In this study, a novel intramolecular DNAzyme assembly method was developed for highly specific miRNA analysis. Compared to traditional DNAzyme-based assembly methods, this approach showed significantly improved analytical performance in terms of dynamic range, assay sensitivity, and speed. The method demonstrated specific detection of miRNA with a remarkably low detection limit.