DNA-only, microwell-based bioassay for multiplex nucleic acid detection with single base-pair resolution using MNAzymes

In disease diagnostics, single- and multiplex nucleic acid (NA) detection, with the potential to discriminate mutated strands, is of paramount importance. Current techniques that rely on target amplification or protein-enzyme based signal amplification are highly relevant, yet still plagued by diverse drawbacks including erroneous target amplification, and the limited stability of protein enzymes. As a solution, we present a multicomponent nucleic acid enzymes (MNAzymes)-based system for singleplex and multiplex detection of NA targets in microwells down to femtomolar (fM) concentrations, without the need for any target amplification or protein enzymes, while operating at room temperature and with single base-pair resolution. After successful validation of the MNAzymes in solution, their performance was further verified on beads in bulk and in femtoliter-sized microwells. The latter is not only a highly simplified system compared to previous microwell-based bioassays but, with the detection limit of 180 fM, it is to-date the most sensitive NAzyme-mediated, bead-based approach, that does not rely on target amplification or any additional signal amplification strategies. Furthermore, we demonstrated, for the first time, multiplexed target detection in microwells, both from buffer and nasopharyngeal swab samples, and presented superior single base-pair resolution of this assay. Because of the design flexibility of MNAzymes and direct demonstration in swab samples, this system holds great promise for multiplexed detection in other clinically relevant matrices without the need for any additional NA or protein components. Moreover, these findings open up the potential for the development of next-generation, protein-free diagnostic tools, including digital assays with single-molecule resolution.