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

Lin, Ya-Hui; Chang, Kung-Yao

RNAs were synthesized by in vitro transcription from appropriate DNA templates cloned into PUC-18 using T7 RNA polymerase. The purified RNAs were dephosphorylated by calf intestine alkaline phosphatase (Roche) and 32 P -labeled at the 5 -end using T4 polynucleotide kinase (NEB) in the presence of [␥ -32 P] ATP. In-line probing assays were performed following published protocols (26, 27) . Briefly, approximately 30 000 CPM per reaction of 5 32 P-labeled RNAs were incubated with varied amounts of theophylline (0-1 mM for final concentration) in in-line probing reaction buffer (50 mM Tris-HCl, pH8.3, 20 mM MgCl 2 , 100 mM KCl) at room temperature for 41 h. Partial alkaline digested RNA ladders were prepared by incubating labeled RNAs in alkaline buffer (50 mM Na 2 CO 3 , 1 mM EDTA, pH 9) at 95 • C for 5 min, while guanine-specific sequencing ladders were obtained by procedures described in next section. All reactions were terminated by adding gel loading buffer (95% Formamide, 18 mM EDTA, 0.025% SDS, 0.025% Xylene Cyanol, 0.025% Bromophenol Blue). Spontaneous RNA cleavage products from in-line probing assays and related markers were separated by 10% denaturing polyacrylamide gel electrophoresis and exposed to a phosphorimager screen after drying of the gels. The phosphorimager screen was scanned by Typhoon FLA 7000 phosphorimager (GE) and the radioactivity of spontaneous RNA-cleavage products was analyzed and quantified by ImageQuantTL software. For loading difference calibration, the quantified intensity values of the cleavage-bands of interest were normalized against value of a band corresponding to residue G13416 of the same lane.