Target-Catalyzed Self-assembly of DNA-Streptavidin Nanogel for Enzyme-free miRNA Assay.

Rapid, sensitive, specific, and user-friendly microRNA (miRNA) assay is highly demanded 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, we present a simple method for miRNA assay based on the one-pot self-assembly of Y-shaped DNAs with streptavidin via a 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 non-specific 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 three-dimensional 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. This article is protected by copyright. All rights reserved.