cOA synthesis

All reactions were carried out in 50 mM Tris HCl pH 8.0, 150 mM NH₄Cl, 5% (v/v) glycerol, 500 µM ATP and 10 mM Mg²⁺ (TNG buffer) at 37°C except where differences in divalent metal or ATP concentrations are noted. Reactions analyzed by PAGE were supplemented with 30 nM α-³²P-ATP 3000 Ci/mmol. Reaction conditions for Figures 3 and ​and44 included 200 nM Cas10-Csm^Csm3D32A, 200 nM target RNA in TNG buffer for 4-h incubation. cOA synthesis was performed overnight to generate the products for mass spectrometry (Fig. 4B). Exonuclease treatment to determine cOA topology was performed as follows: 5 µL cOA reaction product (Fig. 4A) was incubated with 20 units T4 PNK for 1 h at 37°C in a total volume of 10 µL in PNK buffer, T4 PNK was then inactivated at 65°C for 2 min. A total of 2.5 µL NEB Buffer 4 and 10 units exonuclease T (NEB) were added to the reaction, which was then incubated at room temperature for 1 h. Endonuclease treatment of cOA products was performed as follows: 5 µL of cOA reaction product (Fig. 4A) was incubated with 160 units endonuclease S1 in S1 nuclease buffer (Promega) in 10 µL total volume for 1 h at 37°C.

The reactions in Figure 5A were performed with Cas10-Csm 200 nM, 200 nM target RNA or DNA as indicated in TNG buffer for 2-h incubation. Reactions in Figure 5D were performed with 200 nM Cas10-Csm or Cas10-Csm^Csm3D32A, 200 nM ssRNA-01 in TNG buffer. Figure 5E reactions were carried out with 100 nM Cas10-Csm and 100–3200 nM ssRNA-01 in TNG buffer for 60 min. cOA synthesis progress curves (Fig. 6C,D) were obtained using 100 nM Cas10-Csm and 3000 nM ssRNA-01 or Cas10-Csm^Csm3D32A and 3000 nM ssRNA-01 incubated in TNG buffer. cOA synthesis in the presence of S. cerevisiae total RNA was performed with Cas10-Csm 200 nM in TNG buffer for 2-h incubation. ssRNA-01 was present at 200 nM where indicated. S. cerevisiae total RNA used in Figure 5A was obtained using the Ambion RiboPure Yeast kit and added to reactions to 480 µg/mL as indicated. For reactions measuring the effect of mismatches on cOA synthesis (Figs. 7, ​,8),8), reactions were carried out with 100 nM Cas10-Csm or Cas10-Csm^Csm3D32A, 400 nM target RNA in TNG buffer for 12 min.

PAGE analysis of cOA reaction products was carried out with 24% w/v acrylamide (19:1), 8 M urea, tris-borate-EDTA gels. Prior to gel loading, cOA synthesis reactions were quenched by adding 1:1 formamide dye and incubating at 70°C for 2 min. Electrophoresis was performed at 2900 V for approximately 3 h.