wt ZTP riboswitch equilibrium refolded with 1 mM ZMP
Randomly biotinylated DNA templates were prepared that consisted of an E. coli promoter followed by DNA encoding the C. beijerinckii pfl Riboswitch. DNA template was incubated with streptavidin after creating open complexes with E. coli RNA polymerase, followed by initiating in vitro transcription with the addition of Mg++. Reactions were run for 30s at 37C in standard transcription buffer. RNAs were then purified, denatured and equilibrated before probing with BzCN, or a control solvent (DMSO and extraction with Trizol. Extracted RNAs were then processed according to the SHAPE-Seq v2.1 protocol for cDNA synthesis and library preparation prior to sequencing (doi:10.1016/j.ymeth.2016.04.002).