Target‐Catalyzed Self‐Assembly of DNA‐Streptavidin Nanogel for Enzyme‐Free miRNA Assay

Abstract Rapid, sensitive, specific, and user‐friendly microRNA (miRNA) assays are in high demand for point‐of‐care diagnosis. Target‐catalyzed toehold‐mediated strand displacement (TMSD) has received increasing attention as an enzyme‐free molecular tool for DNA detection. However, the application of TMSD to miRNA targets is challenging because relatively weak DNA/RNA hybridization leads to failure in the subtle kinetic control of multiple hybridization steps. Here, a simple method is presented for miRNA assay based on the one‐pot self‐assembly of Y‐shaped DNAs with streptavidin via an miRNA‐catalyzed TMSD cascade reaction. A single miRNA catalyzes the opening cycle of DNA hairpin loops to generate multiple Y‐shaped DNAs carrying biotin and a quencher at the end of their arms. Introducing a single base‐pair mismatch near the toehold facilitates RNA‐triggered strand displacement while barely disturbing nonspecific reactions. The Y‐shaped DNAs are self‐assembled with fluorescently labeled streptavidin (sAv), which produces nanoscale DNA‐sAv nanogel particles mediating efficient Förster resonance energy transfer in their 3D network. The enhancing effect dramatically reduces the detection limit from the nanomolar level to the picomolar level. This work proves that TMSD‐based DNA nanogel with a base‐pair mismatch incorporated to a hairpin structure is a promising approach towards sensitive and accurate miRNA assay.