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

Sensitive, simple, and rapid detection of nucleic acid sequences at point-of-care settings is still an unmet quest. Magnetic readout assays combined with toehold-mediated strand displacement-based circuits are amplification- and wash-free, essential features for contributing to this demand. Nevertheless, nonenzymatic strand displacement circuits are slow, with low sensitivity for early disease diagnostics. Here, we propose novel mismatch-assisted toehold exchange (MATE) magnetic cascades, wherein magnetic susceptibility increases by dissociation of magnetic nanoparticles (MNPs) from engineered magnetic clusters upon detection of a nucleic acid target in solution. The MATE relies on the generation of an allosteric toehold by spontaneous dissociation to efficiently recycle the target, amplify magnetic signal output, and enhance the assay's kinetics. We show that introducing a mismatch in the allosteric toehold domain enhances the overall declustering kinetics 7-fold, as also confirmed with oxDNA simulations, with the largest effect gained for the mismatch being closest to where the branch migration by the target ends. By integrating MATE into magnetic diagnostics cascades, we demonstrate similar sensitivity in a 12-fold shorter assay time compared to our previous circuit design. Our work makes a major leap toward bringing MNP-based diagnostics much closer to the clinical point-of-care settings by offering a simple, rapid, isothermal, and nonenzymatic assay workflow.