In vitro RNA Synthesis and Purification

RNAs were prepared by in vitro transcription. Plasmids with the 9S, RprA and GlmZ RNA genes were generously provided by A.J. Carpousis (CNRS, Toulouse), Kai Papenfort (Jena), and Boris Görke (Vienna), respectively. First, genes were amplified by PCR using primers which were also adding T7 promoter. Next, RNA was synthesized from the PCR amplified product using T7 RNA polymerase at 37°C, followed by treating the reaction mixture with TURBO DNase for 15–20 min at 37°C. Finally, the RNA was purified by urea-PAGE followed by electroelution at 4°C and 100V (EluTrap, Whatman) (Bandyra et al. 2018 (link)). In order to generate 5′-monophosphorylated RNA, rGMP was used in addition to rGTP (5:1 molar ratio) while keeping other reaction component and purification steps same as before (Bandyra et al. 2018 (link)). For all RNAs, purity was checked in 8% urea-PAGE gel stained with SYBRgold RNA dye (Thermo Fisher).