Fig. 4. Incorporation of cA₃-activated nucleases into Csm-based RNA detection assay.

a The target bound TtCsm complex primarily generates cA₄ and cA₃. Schematic summarizes cA₄- and cA₃-dependent activities of nucleases biochemically tested. N/D – not detected; Asterisks (*) indicate nucleases that have sequences preferences (Supplementary Fig. 6). b CtNucC (15 nM) is activated by cA₃ (20 nM) and cleaves plasmid DNA into short fragments in 15 min. c Deep sequencing of DNA fragments generated after 5 min of incubation with CtNucC revealed the preferential cleavage sites (ANNT). The reduced sequencing depth at the cut site is consistent with a cleavage mechanism producing 3′-overhangs that are removed by T4 DNA polymerase when sequencing library is prepared. d CtNucC (300 nM) cleavage assay with fluorescent dsDNA reporter across eight concentrations of cA₃ (shown with colors). Data is shown as mean (center line) of four replicates ± S.D. (ribbon). e–g TtCsm RNA detection assays coupled with CtNucC (dsDNA reporter that include preferred cleavage sequence), AaCan2 (ssRNA reporter), and combination of AaCan2 and CtNucC (both reporters). Reactions were performed using samples with target RNA concentrations ranging from 10⁷ to 10⁴ copies/µL. Samples were prepared by spiking IVT fragment of SARS-CoV-2 N gene in total RNA of SARS-CoV-2 negative nasal swab. Cleavage of the fluorescent reporter was detected by measuring fluorescence every 10 s in a real-time PCR instrument. Simple linear regression was used to determine slopes for four replicates. See Supplementary Fig. 13 for fluorescent curves used in the analysis. Data were plotted as mean (n = 4) ± S.D. and analyzed with one-way ANOVA. All samples were compared to the non-target RNA control (NTC) using one-tailed post-hoc Dunnett’s test. ***p < 0.001; **p < 0.01; *p < 0.05. AU – arbitrary units. Source data are provided as a Source Data file.