High-speed AFM imaging of the Entropic Disassembly of DNA Origami for Single-Molecule Biosensing

Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis, but their small size and instability hinder direct detection. The detection of small RNA, such as miRNA, using solid-state nanopores typically involves the binding of miRNA to a larger carrier molecule to generate signals. However, this approach is entropically unfavourable, and it is prone to RNAse degradation in the environment leading to accidental digestion of miRNA prior to analysis. Here, we present an alternative approach based on DNA origami disassembly driven by toehold mediated strand displacement (TMSD). Symmetrical DNA origami dimers are separated into monomers via TMSD using miRNAs as invading strands. We visualized the real-time dynamics of dimer separation at high resolution using high-speed atomic force microscopy (HS-AFM), providing direct insight into the kinetics of the TMSD process. The entropically driven disassembly process can be followed using single molecule nanopore sensing leading to a quantitative readout of the ratio of dimers to monomers. This direct read-out method enabled the multiplexed detection of miRNAs. Due to the near irreversible process of the TMSD, we performed the detection of miRNA in crude RNA tissue extracts under the presence of RNAse and showed that our approach allowed the robust detection of small RNA that is unaffected by the complex degrading environment.