Figure 2. Approximately 1000 molecules cA₄ are made per molecule of RNA target.

(A) Upper panel shows phosphorimages of thin-layer chromatography of cyclic tetra-adenylate (cA₄) made by S. solfataricus (Sso) Csm complex (470 nM carrying the CRISPR RNA A26) across a range of RNA target concentrations (0.1, 1, 10, 25,100 nM) complementary to the A26 CRISPR RNA at 70°C. Lower panel shows cA₄ synthesised with a cleavage resistant (phosphorothioate) form of the RNA target. (B) Bar graph of the concentration of cA₄ generated with increasing cleavable and cleavage-resistant RNA target generated by quantifying the densiometric signals from A, with an α-³²P-ATP standard curve (Figure 2—figure supplement 1). Error bars indicate the standard deviation of the mean of three technical replicates, with individual data points shown as clear circles. No data are shown for 1 nM cleavable RNA target as cA₄ generated was below detection limits. (C) Bar chart quantifying the number of molecules of cA₄ generated per molecule of cleavable or cleavage resistant target RNA across a range of RNA target concentrations. On average SsoCsm synthesised 980 ± 24 and 3100 ± 750 molecules of cA₄ per molecule of cleavable and cleavage resistant target RNA, respectively. (D) Cartoon depicting the cellular implications of ~1000 molecules of cA₄ generated per molecule of RNA target, which in S. solfataricus would equate to ~6 µM cA₄ within the cell.

Figure 2—figure supplement 1. Example of ATP standard curve used to determine the concentration of ATP converted to cyclic tetra-adenylate (cA₄).

Left-hand side panel shows duplicate serial dilution of ³²P-α-ATP (5 nM) and ATP (500 µM) mix spotted (1 µl) on a thin-layer chromatography (TLC) plate. The right-hand side panel is a plot of the densiometric signal quantified from the TLC plate after phosphorimaging. The mean densiometric signal is plotted and errors bars showing the standard deviation are plotted but not visible due to their scale. The densiometric signal corresponding to cA₄ was compared to the standard curve to determine the concentration of ATP converted. Duplicate standard curves were carried out for each replicate assay examining cA₄ synthesis.

Figure 2—figure supplement 2. Rate of cA₄ synthesis by S. solfataricus type III-D effector complex.

Plot showing the concentration of cA₄ generated by SsoCsm (~4.2 µM) over time in the presence of 25 nM RNA target, 0.5 mM ATP and 2 nM ³²P-α-ATP at 70°C. The data were originally reported in Rouillon et al. (2018) and here were fitted to an exponential equation to determine the kinetic rate constant of cA₄ synthesis (0.04 ± 0.01 min⁻¹). Error bars show the standard deviation of the mean of three technical replicates.