6230 tofms

The cOA synthesis assays were performed as previously described^{10 (link)}. In brief, a mixture containing 160 μCi [α-³²P]-ATP (PerkinElmer) and 500 μM ATP was incubated with 100 nM LlCsm complex, 200 nM target RNA at 37 °C overnight in a cOA synthesis buffer containing 33 mM Tris-acetate pH 7.6 (at 32 °C), 66 mM potassium acetate, 10 mM MgCl₂. The reaction products were heat-denatured at 95 °C for 10 min and centrifuged at high speed. The supernatant was mixed with formamide dye, resolved by 8 M Urea, 24% PAGE gels in 1× TBE running buffer at 80 V for 240 min. The gels were carefully sandwiched between non-porous cellophane sheet and a porous gel drying sheet and dried for 120 min, developed for 30 min using Phosphor Screen (GE Healthcare Life Science), and visualized using the Typhoon Gel Imaging System (GE Healthcare Life Science).
To determine which cOAs are produced by LlCsm by mass spectrometry, the same reaction was carried out without [α-³²P]-ATP. The reaction products were heat-denatured at 95 °C/10 min and centrifuged at high speed. The supernatant was analyzed by mass spectrometry on an Agilent 6230 TOF-MS with the Agilent Mass Hunter Workstation Software TOF 6500 series in positive ion mode. Spectrum was analyzed using Agilent Mass Hunter Qualitative Analysis Navigator v.B.08 and visualized using GraphPad Prism.

The cell suspension (3 mL) was acidified to pH 4.0 with HCl prior to extraction with 6 mL of ethyl acetate. The ethyl acetate extracts were kept at room temperature for 30 min with gentle shaking prior to centrifugation at 4 °C and 4000 rpm for 10 min. The ethyl acetate layer was transferred to new microcentrifuge tubes and evaporated with the aid of a Speed Vac Concentrator. Dried samples were resuspended in 300 µL of methanol and stored at −20 °C. Quantitative determination was carried out by LC-MS using an Agilent 6230 TOF Ms (see above) in the positive ion mode. Samples were separated in a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 μm particle size, 120 Å pore size; Agilent) using a flow rate of 0.4 mL/min and the following gradient: 95% A (2 min), 40% A (11 min), 5% A (26 min), and reequilibration to initial conditions (11 min); A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile. Phenazines were detected using full scan mode (m/z 130 to 350) and quantitated by measuring the integrated peak area of the corresponding [M + H]⁺ ions and a standard curve constructed using commercially available PCA (Evo Blocks Ltd., Budapest, Hungary) and PYO (Cayman Chemical, Ann Arbor, MI, USA).