Velos pro

Unlabeled cOA production assays were performed as described above except the reactions were incubated for 2 h. Liquid chromatography high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system as previously described (41 (link)).

Cyclic oligoadenylate production was triggered by adding 2 μM crude target RNA to 0.8 μM Csm, 1 mM adenosine triphosphate (ATP), 5 mM MgCl₂, 0.1 U μl⁻¹ SUPERase•In™ (Thermo Scientific), 50 mM Tris–HCl, 50 mM NaCl, pH 8.0, and incubating at 30°C for up to 2 h. The reaction was stoped by phenol–chloroform–isoamyl alcohol extraction. Reaction mixtures that were used for Csm6 or Tsu Csx1 activation assays were further extracted with chloroform–isoamyl alcohol to remove residual phenol. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system. Samples were desalted on C18 cartridges (Harvard Apparatus). Compounds were separated on a Kinetex 2.6 μm EVO C18 column (2.1 × 5 mm, Phenomenex) using the following gradient of acetonitrile (B) against 20 mM ammonium bicarbonate (A): 0–2 min 2% B, 2–10 min 2–8% B, 10–11 min 8–95% B, 11–14 min 95% B, 14–15 min 95–2% B, 15–20 min 2% B at a flow rate of 350 μl min⁻¹ and column temperature of 40°C. UV data were recorded at 254 nm. Mass data were acquired on the FT mass analyser in positive ion mode with scan range m/z 200–2000 at a resolution of 30 000.

Samples were generated by incubating Csx3 (10 μM dimer) with 100 μM synthetic cA₄ (BIOLOG Life Science Institute, Bremen) in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT and 2 mM MnCl₂ for 10 min at 50°C. Reactions were quenched by adding EDTA to a final concentration of 25 mM and acidified with trifluoroacetic acid. The acidified sample was bound to a C18 cartridge (Harvard Apparatus), and salts and buffer were washed away with 0.1% trifluoroacetic acid, 2% acetonitrile. Adenylates were eluted from the cartridge with 20 mM ammonium bicarbonate, 50% acetonitrile leaving most protein bound to the resin. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system as previously described (Grüschow et al., 2019 (link)). Data were analysed using Xcalibur (Thermo Scientific).