Pnk buffer a

RNAs were in vitro transcribed and purified as described previously (Lahr et al., 2015 (link)). For studying the un-capped RNAs, transcribed RNAs were treated with alkaline phosphatase (Roche Life Sciences (Indianapolis, IN), cat. no. M183A) and 5’ end-radiolabeled with [γ-³²P]-ATP. To generate capped RNAs, the 5’ triphosphate required for the capping reaction was regenerated in TOP RNAs by incubation of 50 nM RNA with T4 PNK in PNK buffer A (ThermoFisher Scientific cat. no. EK0032) with 3 mM ATP for 20 min at 37°C followed by addition of 5 units of nucleoside monophosphate kinase (Roche Life Sciences, cat. no. 10107948001). RNA was purified by phenol-chloroform extraction, MicroSpin G-25 desalting columns (GE Healthcare Life Sciences (Marlborough, MA), cat. no. 27-5325-01), and ethanol precipitation. RNAs with a 5’ triphosphate were subsequently capped and radiolabeled using vaccinia capping enzyme (NEB (Ipswich, MA), cat. no. M2080S) and [α-³²P]-GTP or GTP according to the manufacturer’s protocol.

To facilitate the measurement of the product formation by gel electrophoresis, all the reactions were doped with minor amounts (0.01 μM) of radioactively labeled W or WXY fragment (γ³²P). These radioactively labeled RNAs were prepared by mixing 1 μM of RNA with 1X of Shrimp Alkaline Phosphatase buffer (rSAP reaction buffer, New England BioLabs), 1 U/μL of rSAP enzyme (New England BioLabs, Product no.: M0371S), and water in 10 μL reaction scale. Samples were incubated at 37 °C for 40 min and enzyme was heat inactivated at 70 °C for 10 min. The SAP reaction samples were then directly used for kinase reaction by mixing 0.5X of polynucleotide kinase buffer (PNK Buffer A, Thermo Scientific), 5 μL of γ³²P-ATP (10 mCi/mL, BRIT Hyderabad, India, Product No.: PLC 101), 0.4 U/μL PNK enzyme (Thermo Scientific, Product No.: EK0031) and water in 20 μL reaction scale. The samples were incubated at 37 °C for 1 h and the reaction was stopped by adding an equal volume of gel-loading buffer (90% formamide, 0.01% xylene cyanol, 0.01% bromophenol blue). Labeled RNAs were purified on 12% denaturing polyacrylamide gels, eluted in 0.3 M of sodium acetate pH 5.5 (2 h at 37 °C) followed by isopropanol precipitation as mentioned above. The pelleted RNAs were resuspened in water and used for the RNA catalysis experiments.