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

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Circulating microRNAs (miRNAs) are valuable disease biomarkers.
• Current miRNA detection methods using nanopores are hindered by miRNA instability and RNase activity.
• Carrier molecules used in nanopore detection can be degraded by RNases, leading to false negatives.

Purpose of the Study:

• To develop a robust method for detecting circulating microRNAs (miRNAs) resistant to RNase degradation.
• To utilize DNA origami and toehold-mediated strand displacement (TMSD) for sensitive and specific miRNA detection.
• To demonstrate multiplexed miRNA detection in complex biological samples.

Main Methods:

• Designed a symmetric DNA origami dimer that disassembles into monomers via TMSD.
• Used miRNAs as invading strands to trigger TMSD-driven dimer separation.
• Visualized real-time TMSD dynamics using high-speed atomic force microscopy (HS-AFM).
• Employed single-molecule nanopore sensing for quantitative endpoint analysis of dimer-to-monomer ratios.

Main Results:

• Successfully visualized the nanoscale mechanical dynamics of TMSD using HS-AFM.
• Achieved quantitative endpoint analysis of dimer disassembly via single-molecule nanopore sensing.
• Demonstrated multiplexed detection of miRNAs.
• Successfully detected miRNAs in crude RNA tissue extracts despite RNase presence, showcasing robustness.

Conclusions:

• The DNA origami disassembly approach driven by TMSD offers a robust method for small RNA detection in degrading environments.
• This technique overcomes the limitations of RNase sensitivity in current miRNA biomarker detection strategies.
• The combination of HS-AFM and nanopore sensing provides powerful tools for studying nanoscale molecular dynamics and enabling sensitive biomarker detection.