Refolded synthetic TAR (nucleotides 18–44) was radioactively labeled with 32P-γ–ATP using T4-polynucleotide kinase. A 10-µl reaction was prepared with 200 nM TAR, 0.3 mCi 32P-γ–ATP (7000 Ci/mmol, MP Biomedicals, Sohon, OH), and 10 units of T4-polynucleotide kinase (New England BioLabs, Ipswich, MA) in 70 mM Tris/HCl pH7.6, 10 mM MgCl2, 2 mM DTT. After incubating at 37°C for 1 hr, 25 µl of annealing buffer (20 mM Na HEPES pH 7.3, 100 mM KCl, 3 mM MgCl2) were added to the reaction. The mixture was purified twice over Illustra G25 spin columns (GE Healthcare, Piscataway, NJ) to remove free nucleotides. The purified labeled TAR was diluted to 10 nM (3000–5000 cpm/ µl) with annealing buffer for storage and use in EMSAs.
Binding reactions (10 µl) were carried out in 20 mM Na HEPES pH 7.3, 100 mM KCl, 3 mM MgCl2, 1 mM DTT, 4% glycerol with 12 units RNasin (Promega, Madison, WI), 10 µg/ml BSA, and 5 µg/ml poly(I:C) (Invivogen, San Diego, CA). Each reaction contained 100 pM labeled TAR RNA. Reactions were incubated at 20°C for 30 min and RNA-binding complexes were separated on a pre-run 6% polyacrylamide gel in 0.5x TBE (100 V, 1 hr at 4°C). Gels were dried, exposed to storage phosphor screens, and measured on a Typhoon phosphorimager (GE Healthcare, Piscataway, NJ).
Binding reactions (10 µl) were carried out in 20 mM Na HEPES pH 7.3, 100 mM KCl, 3 mM MgCl2, 1 mM DTT, 4% glycerol with 12 units RNasin (Promega, Madison, WI), 10 µg/ml BSA, and 5 µg/ml poly(I:C) (Invivogen, San Diego, CA). Each reaction contained 100 pM labeled TAR RNA. Reactions were incubated at 20°C for 30 min and RNA-binding complexes were separated on a pre-run 6% polyacrylamide gel in 0.5x TBE (100 V, 1 hr at 4°C). Gels were dried, exposed to storage phosphor screens, and measured on a Typhoon phosphorimager (GE Healthcare, Piscataway, NJ).