Multi-Amplified Sensing of MicroRNA by a Small DNA Fragment-Driven Enzymatic Cascade Reaction

Combining technological developments such as nanomaterials, DNA nanotechnology, and functional enzymes has great potential to facilitate next generation high performance molecular diagnostic systems. In this work, we describe a microRNA (miRNA) detection assay that combines target recycling and isothermal amplification in an elegantly designed enzyme-mediated cascade reaction. Target recycling is driven by the action of duplex-specific nuclease (DSN), resulting in highly amplified translation of input miRNA to short output DNA fragments. These fragments act as highly specific initiators of rolling circle amplification (RCA), an isothermal reaction that outputs a large volume of polymeric DNAzymes per initiator, and finally a fluorogenic output signal. Based on careful electrophoretic analysis we observed that the DSN produces ca. 10 nt DNA fragments from DNA/miRNA duplexes, regardless of the length of DNA strands. Target recycling yielded ca. 5 orders of magnitude amplification through the DSN-assisted recycling system on magnetic particles, and the RCA yielded a further 2 orders of magnitude. The final assay exhibited a limit of detection of 1.8 fM of miRNA spiked into 20% human serum, and showed excellent selectivity for miR-21 versus single base-mismatched sequences and other cancer-related miRNAs. The developed assay was further employed to determine accurate amounts of miR-21 in total RNA samples extracted from human cancer cell lines and normal cells, confirming the applicability of the assay for direct and absolute quantification of mature specific miRNA in real biological samples.