A general strategy to inhibiting viral -1 frameshifting based on upstream attenuation duplex formation

Hu, Hao-Teng; Cho, Che-Pei; Lin, Ya-Hui; Chang, Kung-Yao

The purified RNA transcripts were treated with rAPid alkaline phosphatase (Roche) in the presence of RNase inhibitor (Promega) at 37 • C for 1 h to remove the unlabeled 5 -phosphate. After the inactivation of phosphatase by incubation at 75 • C for 2 min, 32 P -␥ ATP (Amersham) and T4 polynucleotide kinase (Roche) were added and the reaction continued for 40 min at 37 • C. The 32 P -labeled RNAs were purified by 20% denaturing polyacrylamide gel and recovered by crush and soak procedures. After ethanol precipitation, the labeled RNAs were recovered by centrifugation. To analyze RNA-DNA interactions, labeled RNA probes (10 000 CPM per reaction) were incubated with various amounts of antisense DNA in a final volume of 10 l of 1× TBE buffer containing 100 mM NaCl and 0.1 mM EDTA. The RNA-DNA complexes were heated at 80 • C and annealed for 30 min at 30 • C. The reactions were then mixed with 2 l of 40% sucrose as the loading buffer and loaded into a 20% non-denaturing polyacrylamide gel (19:1 acryl:bisacryl ratio) in 0.5× TBE (Tris-boric acid-EDTA) run at a constant voltage of 150V at 4 • C for EMSA analysis. The results were visualized by autoradiography using a Typhoon FLA7000 phosphorimager (GE).