Fig. 4: Cir-mediator-induced Cas12a autocatalysis amplifies trans-cleavage in AutoCAR-1 scheme. | Nature Communications

Fig. 4: Cir-mediator-induced Cas12a autocatalysis amplifies trans-cleavage in AutoCAR-1 scheme.

From: Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing

Fig. 4

A The AutoCAR-1 scheme for amplified ssDNA cleavage using ssDNA-linked fluorescence-quenched reporters. B The fluorescence signal intensity differences between reporter trans-cleavage in a standard CRISPR/Cas12a and the AutoCAR-1 systems measured by the fluorescent signal produced by cleavage of fluorescent quenched ssDNA reporters (n = 3 independent reactions). In comparison with a standard Cas12a catalytic system without Cir-mediators, the reporter trans-cleavage increased 7.3 times over 3600 s (60 min) and 14.4 times over 7200 s (120 min) reactions at room temperature. (Method 7). C The reporter trans-cleavage in AutoCAR-1 increased with increasing Cir-mediator concentration (P300(0-5nM) = 0.0046, P600(0-5nM) = 0.0016, P600(5-25nM) < 0.0001, n = 3 independent reactions). (Method S6). D Time dependence of the fluorescence signal for AutoCAR-1 in comparison to a standard CRISPR/Cas12a reaction (n = 3 independent reactions). Our data show differences in reporter trans-cleavage kinetics in a standard Cas12a catalytic system (linear trend, y = 0.014x + 9.1129, goodness of fit R2 = 0.9584) and Cir-mediator-assisted Cas12a autocatalysis reaction (super-linear trend, exponential fit, y = 12.102e0.0023x, goodness of fit R2 = 0.9844). These results indicate that instead of the well-established linear trend14, AutoCAR-1 produces a nonlinearly increasing signal (Method 7). E The influence of different concentrations of reporters in the AutoCAR-1 system with 50 nM Cir-mediators (n = 3 independent reactions) (Method 8). F Magnified section of Fig. 4E showing the time window 1 (0–6 min). G Magnified section of Fig. 4E showing the time window 2 (20–30 min). H The influence of different concentrations of Cir-mediators in the AutoCAR-1 system with excess reporters (n = 3 independent reactions, Method 9). I Magnified section of Fig. 4H showing the time window 1 (0–6 mins). J Magnified section of Fig. 4H showing the time window 2 (20–30 min). (Two tails student T test, Error bars represent mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.001, a.u = arbitrary units).

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