Chemical probing of U2 snRNA - Capillary electrophoresis chromatograms

1m7 chemical probing of the synthetic RNA consisting of the first 100 nucleotides of U2 snRNA along with a series of mutations in the Stem I region and data analysis was carried out essentially as described previously in "Kladwang W, VanLang CC, Cordero P, Das R (2011) A two-dimensional mutate-and-map strategy for non-coding RNA structure. Nat Chem 3: 954-962". 

Briefly synthetic U2 snRNA templates flanked by two stable stem-loops to use as probing standards and a 3′ tail for primer extensions were generated by PCR from overlapping oligonucleotides and used in T7 run-off transcription reactions. The transcribed RNA was separated on a 5% (v/v) denaturing polyacrylamide gel. Correlating bands identified by UV shadowing were excised, and the RNA was isolated from excised bands by soaking gel slices in 0.3 M sodium acetate pH 4.8, 1 mM EDTA, 10% phenol overnight followed by ethanol precipitation.

For chemical probing, 0.6 pmol of RNA in 50mM Na-HEPES was heated to 95 ˚C for 3 minutes and allowed to fold at room temperature for 20 minutes followed by the addition of 10 mM magnesium chloride. The RNA was aliquoted into a 96-well plate with 5 mM 1m7 or water and incubated for 10 minutes at room temperature followed by the addition of oligo dT magnetic beads (Ambion) in 0.25 M Na-MES pH 6.0, 1.5 M NaCl, and 6.4 nM FAM-labeled primer. RNA/primers were isolated by magnetic bead immobilization, washed twice with 70% ethanol, and resuspended in 2.5 μL water.

For reverse transcription, 20 units SuperScript III reverse transcriptase (Thermo Fisher Scientific), 5 mM dithiothreitol, 0.8 mM dNTPs, 50 mM Tris-HCl pH 8.3, 75 mM potassium chloride, and 3 mM magnesium chloride was added, and reactions were incubated for 30 minutes at 48˚C. Ladders were generated by the addition of ddNTPs to additional control reactions. Following reverse transcription, 0.2 M sodium hydroxide was added, and samples were incubated for 3 minutes at 90˚C to degrade the remaining RNA. Samples were neutralized by the addition of 1.4 M sodium chloride and 0.6 M hydrogen chloride, followed by 1.3 M sodium acetate pH 5.2. DNA was purified by magnetic bead immobilization, washed twice with 70% ethanol, and resuspended in Hi-Di formamide supplemented with R0X350 dye standards (1:8 ROXF) (Thermo Fisher Scientific). 1:3 and 1:15 dilutions of the samples in ROXF were analyzed by capillary electrophoresis (ELIM Biopharmaceuticals).