Mismatch-Assisted Toehold Exchange Cascades for Magnetic Nanoparticle-based Nucleic Acid Diagnostics

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Diagnostics

Background:

• Point-of-care nucleic acid detection requires sensitive, simple, and rapid methods.
• Existing magnetic readout assays with toehold-mediated strand displacement are amplification- and wash-free but suffer from slow kinetics and low sensitivity.
• Nonenzymatic strand displacement circuits are particularly slow, hindering early disease diagnostics.

Purpose of the Study:

• To develop a novel, rapid, and sensitive nonenzymatic nucleic acid detection method for point-of-care applications.
• To enhance the kinetics and signal output of magnetic readout assays using toehold-mediated strand displacement.
• To improve the efficiency of target recycling and magnetic signal amplification in diagnostic assays.

Main Methods:

• Proposed novel mismatch-assisted toehold exchange (MATE) magnetic cascades.
• Engineered magnetic clusters that dissociate upon target nucleic acid detection, increasing magnetic susceptibility.
• Utilized spontaneous dissociation to generate an allosteric toehold for target recycling and signal amplification.
• Integrated MATE into magnetic diagnostics cascades for enhanced assay performance.
• Employed oxDNA simulations to confirm the effect of mismatches on declustering kinetics.

Main Results:

• MATE cascades demonstrated efficient target recycling, amplified magnetic signal output, and enhanced assay kinetics.
• Introducing a mismatch in the allosteric toehold domain increased declustering kinetics 7-fold.
• The largest kinetic enhancement was observed when the mismatch was closest to the branch migration end.
• MATE integrated into magnetic diagnostics achieved similar sensitivity to previous designs but in a 12-fold shorter assay time.
• Demonstrated a simple, rapid, isothermal, and nonenzymatic assay workflow.

Conclusions:

• MATE magnetic cascades offer a significant improvement in speed and efficiency for nucleic acid detection.
• The developed assay is suitable for point-of-care settings, addressing the unmet need for rapid diagnostics.
• This work advances magnetic nanoparticle-based diagnostics toward clinical applications by providing a faster, simpler workflow.