Cleavage-gated Cas13-to-DNA nanostructure transduction for target-preamplification-free viral RNA detection.

CRISPR-Cas13a enables programmable RNA recognition without target nucleic-acid preamplification, but conventional assays encode collateral RNase activity by cleaving fluorescent reporters. Here, we developed a cleavage-gated strategy that converts transient Cas13a cleavage into a sequence-defined, strand-displacement-competent DNA actuator, shifting signal encoding from cumulative reporter intensity to a discrete scaffold topology. A chimeric masking hairpin (CMH) links Cas13a to a DNA nanostructure sensor (DNA-NS) by using a cleavable RNA loop to constrain a DNA-key bearing a pre-exposed toehold. Target-activated Cas13a cleaves the loop, converting the partially masked key into a strand-displacement-competent intermediate that reconfigures an M13mp18 scaffold from a linear to a looped state, producing a distinct electrophoretic mobility signature. Screening of 40 CMH-sensor-handle combinations identified a 4-nt toehold paired with an 18-nt sensor handle as the best-performing configuration within the tested design space (component-level ΔΔG = -1.19 kcal/mol at 37 °C). Using pooled crRNAs targeting the S, M, and L genomic segments of severe fever with thrombocytopenia syndrome virus (SFTSV), the assay detected the lowest tested input concentration of 10.38 fM in all three replicates. In a retrospective case-control evaluation of 87 plasma-derived RNA samples, the assay agreed with RT-qPCR for 45 of 47 positive specimens and 40 healthy controls, yielding a positive percent agreement of 95.7% and a negative percent agreement of 100%. Replacing the crRNAs redirected recognition to Hantaan virus reference RNA without redesigning the CMH or DNA-NS. This work establishes a modular framework for topology-resolved viral RNA detection without target preamplification.