SHAPE-Seq of Faecalibacterium prausnitzii
The ligand-dependent acylation of the Fpr preQ1-III riboswitch was probed by SHAPE-seq (54). Nicotinic acid imidazole (NAI) was purchased from Sigma-Aldrich (St. Louis). To ensure proper RNA folding and to maintain a control region of constant acylation, we used a cassette with folded 5- and 3-ends as described (1,2). Purified RNA from in vitro transcription was heated in metal-free water for 2 min at 95 C, then flash-cooled on ice. A 3 SHAPE buffer [0.333 M HEPES (pH 8.0), 0.02 M MgCl2, 0.333 M NaCl] was added and the RNA was equilibrated at 37 C for 10 min. A total of 1 uL of preQ1 stock (4 KD in 1 PBS) was added to the RNA. The RNA was incubated at 37 C for 15 min. To this mixture, 1 uL of 10 NAI stock in DMSO (+), or DMSO alone (), was added to a final concentration of 0.06 M. The NAI reaction proceeded for 10 min followed by stopping the reaction using a reaction stop mix comprising 90 uL water, 5 uL 4 M NaCl, 1.5 uL GlycoBlue and 2 uL of 100 mM EDTA pH 8.0 that that contains ethanol for precipitation of the pellet. The sample was precipitated at -20 C for 16 h. The samples were spun down and the pellet was washed using 70% ethanol. The pellet was dried on the benchtop and resuspended in 0.5 TE buffer. Modification of extracted RNA was analyzed using the SHAPE-seq v2.1 workflow as described (3) with an additional purification step where samples were size selected to 160300 bp using a 2% agarose gel on the PippinHT system (Sage Science, MA) as described (4). Each sample was analyzed with SPATS version 1.9.30 (3,5). SHAPE reactivities were calculated for the WT, A52G, A84G, d84, and U8C/A85G.