Fig. 3

Faster target activation accounts for better performance of Ts in RPA-Cas reaction at 25 °C (a). Schematic of assay measuring steady-state cleavage of trans-substrate by RNPs pre-activated with target. At t = 0, RNPs pre-activated with target are reacted with trans-substrate, and the resulting linear accumulation of product enables determination of steady-state trans-substrate cleavage rates. (Bottom) Schematic time courses for two RNPs showing that increase in steady-state rate of trans-substrate cleavage increases rate of product accumulation. (b). Schematic of assay measuring the time course of target-activated trans-substrate cleavage by RNPs. At t = 0, RNPs are reacted with a mixture of activating target and trans-substrate, and the resulting accumulation of product enables determination of rates for both enzyme activation and steady-state trans-substrate cleavage. (Bottom) Schematic time courses for two RNPs (solid red and blue lines) under differing scenarios: (left) identical steady-state rates of trans-substrate cleavage but differing activation rates; (right) identical activation rates but differing steady-state rates of trans-substrate cleavage. The lag phase represents the target-induced activation of RNP nuclease enabling cis- and trans-cleavage, and the subsequent linear phase represent steady-state cleavage of trans-substrate by the activated RNP-target complex. Dashed lines indicate steady state for cleavage of trans-substrate, whose slopes provide rates of cleavage (k_ss), and when back extrapolated intersect the x-axis at the activation time (1/k_act), indicated by vertical grey lines. Increases in rates of either enzyme activation (left) or steady-state trans-substrate cleavage (right) increase rates of product accumulation. (c) Steady-state cleavage of trans-substrate FQ-C₁₀ by RNPs pre-reacted with activating target was conducted at varied temperatures, as depicted in (a), in RNP assembly buffer. Bars represent mean (± SE) steady-state turnover (k_ss) of trans-substrate calculated from slopes of time courses (Supplementary Fig. 3a) normalized to the concentration of activating target serving as a proxy for activated enzyme. Error bars are too small to be visible. (d). Steady-state cleavage of trans-substrate FQ-C₅ by RNPs pre-reacted with target was conducted at 37 °C, as depicted in (a), in RNP assembly buffer or Mg²⁺-supplemented RPA buffer from the TwistDx kit. Bars represent mean (± SE) steady-state turnover (k_ss) of trans-substrate calculated from slopes of time courses (Supplementary Fig. 3b) normalized to the concentration of activating target. In some cases, error bars are too small to be visible. The ratio of rates measured in RPA buffer to RNP assembly buffer is indicated above the bars. (e). Parameters for target-induced activation of trans-substrate cleavage conducted under the activation scheme depicted in (b). Solutions containing Cas12a ortholog RNPs were reacted with a mixture of activating target and trans-substrate FQ-C₅ at 37 °C in Mg²⁺-supplemented RPA buffer from the TwistDx kit. Bars represent mean (± SE) rate of trans-nuclease activation (k_act) or steady-state turnover (k_ss) of trans-substrate calculated from time courses (Supplementary Fig. 3c). In some cases, error bars are too small to be visible. (f). Time courses for target-induced activation of trans-substrate cleavage conducted under the activation scheme depicted in (b). Reactions containing wild-type Ts and LbCas12a and trans-substrate FQ-C₅ were performed at 37 °C or 25 °C in Mg²⁺-supplemented RPA buffer from the TwistDx kit, using an activating target. Symbols represent mean (± SD) reporter values for replicates (n = 3) after subtraction of signals recorded in the absence of target. Open symbols represent data in the linear range. Solid diagonal lines represent linear fitting of steady-state phase used to calculate activation parameters (Supplementary Fig. 5a).