Rsap enzyme

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.