Fig. 4: Cas3 ssDNA cleavage preferences for optimizing probes for CONAN.

A Collateral cleavage activity of EMX1 target sequences using RNA–DNA hybrid probes composed of two deoxynucleotides, and the heat map of the collateral cleavage activity of the two base combinations. B The signal-to-noise ratio in the presence of non-specific cleavage activity without the target. C Evaluation of the collateral cleavage activity with different lengths of adenine and cytosine repeat sequences. D Assessment of the optimal probe sequence with high signal-to-noise ratio by evaluating the collateral cleavage activity of six-base ssDNA probes with various combinations of four deoxyribonucleotides (see also Fig. S7). In A to D, 100 nM of 60-bp oligo DNA was used. E Evaluation of the collateral cleavage activity for MPXV targets with several optimized probes. MPXV 100-bp oligo DNA fragments at 1 nM, 10 nM, and 100 nM were used as templates. For each probe, a calibration curve was created by plotting the target DNA concentration against the RFU values. The relative detectability of each probe was compared by assessing how much lower concentrations of the target could be detected compared to the GAPDH probe. The performance ratio (PR) was defined as the DNA concentration required to reach a given RFU value using the GAPDH probe, divided by the DNA concentration required to reach the same RFU value using the optimised probes. F RPA was performed on 23 fM genomic DNA of the Mpox Zr-599 strain at different incubation times (0, 5, 10, 15, 20, 25, and 30 min) and CONAN activity was measured using improved probes for the RPA products (n = 3). Circle, control probe; triangle, ATATAT probe. #: saturated signals on CFX Connect device.