The precipitate of de-protected RNA (5′-labeled with Dy547) was dissolved in 0.1× PBS to a final concentration of 15 µM, then heated briefly to 95°C and allowed to cool slowly (over 2 h) to room temperature. The resulting RNA was incubated, at a final concentration of 3 µM, in 10 mM KCl, 50 mM Tris, pH 8.5. MgCl2 and peptoid were added to specified concentrations as stock solutions. Reaction mixtures were incubated at room temperature in darkness for 7 d, then precipitated with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. Alkaline hydrolysis was carried out in 10 mM NaHCO3, pH 9.0, at 95°C for 4 min.
ribonuclease T1 digestion was carried out with 0.007 units of ribonuclease T1 (Ambion) in 20 mM Tris, pH 7.15, 50 mM NaCl, 0.1 mM MgCl2, at room temperature for 20 min. The reaction was stopped by addition of 0.2 volumes of 5 mM EDTA and precipitation with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. The precipitates were heated briefly at 95°C in formamide prior to analysis by electrophoresis on a polyacrylamide gel (20%, 19:1 crosslinking, 8 M urea). The gel was visualized by fluorescence (532-nm excitation) with a Typhoon 9200 scanner (Amersham Biosciences) and analyzed with ImageQuant software.
ribonuclease T1 digestion was carried out with 0.007 units of ribonuclease T1 (Ambion) in 20 mM Tris, pH 7.15, 50 mM NaCl, 0.1 mM MgCl2, at room temperature for 20 min. The reaction was stopped by addition of 0.2 volumes of 5 mM EDTA and precipitation with ethanol, sodium acetate, and 5 µg of yeast phenylalanine tRNA. The precipitates were heated briefly at 95°C in formamide prior to analysis by electrophoresis on a polyacrylamide gel (20%, 19:1 crosslinking, 8 M urea). The gel was visualized by fluorescence (532-nm excitation) with a Typhoon 9200 scanner (Amersham Biosciences) and analyzed with ImageQuant software.