Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

Approximately 30 000 CPM per reaction of 5 32 P-labeled Switch-0 or Switch-1 RNAs were denatured at 95 • C for 2 min under different conditions (with or without ligands). The denatured RNAs were refolded on ice for 30 min and then digested with RNase T2 (0.04 U), V1 (0.000125U) or T1 (0.05U) in structure mapping buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl 2 ) at room temperature for 15 min, and stopped by adding gel loading buffer. The RNA alkaline hydrolysis marker was obtained as described above. The guanine-specific and cytosine/uracil sequencing ladders were obtained by denaturing labeled RNAs in RNA sequencing buffer (20 mM sodium citrate pH 5, 1 mM EDTA, 7 M urea) at 55 • C for 5 min followed by RNase T1 (0.02 U) and RNase A (10 −5 ng/l) digestion, respectively. RNase T1 digestion was carried out at room temperature for 15 min or 30 min and RNase A digestion was carried out at room temperature for 3 min. A 5 l aliquot from each reaction was loaded for 10% denaturing polyacrylamide gel electrophoresis and quantified using a similar method to that described for in-line probing assays.