Preparation and Modification of Diverse RNAs

DNA templates (IDT) were synthesized for tRNA^Phe, TPP riboswitch, E. coli 5S, hepatitis C virus IRES domain, T. thermophila group I intron, or O. iheyensis group II intron RNAs in the context of flanking 5' and 3' structure cassettes. Templates were amplified by PCR and transcribed into RNA using T7 RNA polymerase⁴³ . RNAs were purified by denaturing polyacrylamide gel electrophoresis, appropriate regions excised, and RNAs passively eluted from the gel overnight at 4 °C. 16S and 23S rRNAs were isolated from DH5α cells during mid-log phase using non-denaturing conditions^{38 (link)}. For each sample, 5 pM of RNA was refolded in 100 mM HEPES, pH 8.0, 100 mM NaCl, and 10 mM MgCl₂ in a final volume of 10 µL. After folding, RNAs were modified in the presence of 10 mM SHAPE reagent and incubated at 37 °C for 3 min (1M6 and 1M7) or 22 min (NMIA). No-reagent controls, containing neat DMSO rather than SHAPE reagent, were performed in parallel. To account for sequence-specific biases in adduct detection, RNAs were modified using NMIA, 1M7, or 1M6 under strongly denaturing conditions in 50 mM HEPES (pH 8.0), 4 mM EDTA, and 50% formamide at 95 °C. Following modification, RNAs were isolated using either RNA affinity columns (RNeasy MinElute; Qiagen) or G-50 spin columns (GE Healthcare).