Colorimetric detection of viral RNA fragments based on an integrated logic-operated three-dimensional DNA walker

Timely and accurate detection of virus is crucial for preventing spread of disease and early treatment of the infected cases. Herein we design an integrated logic-operated three-dimensional DNA walker for colorimetric detection of viral RNA fragments, by taking SARS-CoV-2 as an example. The DNA walker is composed of small amounts of dually-blocked walking strands and large amounts of dual-stem-loop track strands on gold nanoparticles. The walking strand contains a swing arm domain and a DNAzyme domain blocked at both sides of catalytic core, while the track strand contains a substrate domain located at the peripheral larger loop. Only the presence of both ORF1ab and N RNA fragments can fully de-block the walking strand, which then continuously hybridizes with track strands and cleaves them by DNAzyme-catalyzed hydrolysis. As the cleavage of track strands from long-stranded, double stem-loop structure to short-stranded, linear sequence, the DNA walker shows much lowered stability due to decreased negative charge density and diminished steric repulsion, which then gets aggregated at high salt concentration, accompanied by a visible color change. The colorimetric DNA walker detects RNA fragments down to 1 nM, responds dual viral genes in a "AND" logic way, and shows high specificity to target sequence. It can further detect large nucleic acids containing ORF1ab and N sequences, and reach 200 copies/mL detection limit by coupling a simple upstream amplification of sample. The method may provide a convenient way for reliable detection of viral RNA.