Visualizing and Quantifying microRNA‐Induced DNA Origami Separation at the Nanoscale

Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis, but their small size and instability hinder direct detection. The detection of miRNA using solid-state nanopores typically involves the binding of miRNA to a larger carrier molecule to generate detectable signals. However, these carriers can be affected by RNase activity during sample handling, potentially causing false negatives if the RNA is degraded before nanopore detection. Here, we present an alternative approach based on DNA origami disassembly driven by toehold-mediated strand displacement (TMSD) which can be performed in the presence of RNases. We designed a symmetric DNA origami dimer that undergoes TMSD-driven separation into monomers using miRNAs as invading strands. We visualized the real-time dynamics of dimer separation at high resolution using high-speed atomic force microscopy, directly capturing nanoscale mechanical dynamics of the TMSD process that are inaccessible to ensemble or fluorescence-based measurements. Single molecule nanopore sensing enables quantitative endpoint analysis of dimer separation by measuring the ratio of dimers to monomers. This direct read-out enabled the multiplexed detection of miRNAs. Owing to the near-irreversible nature of TMSD, we detected miRNA in crude RNA tissue extracts in the presence of RNase, demonstrating robust small RNA detection in a complex degrading environment.