Mapping RNA Structural Dynamics

RNA constructs were generated and purified from DNA templates containing a 5′ T7 promoter by fragment-assembly PCR of oligonucleotides (Integrated DNA Technologies)63 (link). RNA was transcribed with T7 RNA polymerase and purified by affinity column (QIAGEN). P5abc RNA (0.2 μM) was folded for 15 min at 37 °C in 0 or 10 mM MgCl₂ and NMR buffer containing 10 mM sodium phosphate and 0.01 mM EDTA (pH 6.4). A pre-denaturation step of the RNA for 2 min at 95 °C did not change the obtained results. RNA was equilibrated to 10 °C for at least 30 min; longer incubations up to 120 min did not change the results for WT P5abc. RNAs were incubated with 4.2 mg ml⁻¹ 1M7 (1-methyl-7-nitroisatoic anhydride) in anhydrous DMSO (final DMSO 5%) for 60 min followed by purification and reverse transcription63 (link). Nucleotides that do not participate in base pairing are selectively modified at the 2′-OH moiety which gives rise to reverse-transcription stops at the modified residues. cDNA fragments were resolved by capillary electrophoresis on a AB 3730 sequencer (Applied Biosystems).