In Vitro Transcription and RNA Purification

All RNA samples were prepared by T7 RNA polymerase (RNAP)-based in vitro transcription following well-established protocols [65 (link)]. In brief, transcriptions were carried out in 40 mM Tris-HCl (pH 8 at 37 °C), 1 mM spermidine, 0.01% Triton-X100, 80 mg/mL polyethylene glycol, 0.3 μM DNA templates (Integrated DNA Technology), 1 mM DTT, 2 U/µL thermostable inorganic pyrophosphatase (New England Biolabs, Ipswich, MA, USA), 5–15 mM ribonucleotide 5′-triphosphates (rNTPs), 5–15 mM MgCl₂, and 0.1 mg/mL T7 RNAP. The reactions were first optimized for appropriate rNTP and MgCl₂ concentrations at the small (50 µL) and medium (500 µL) scales before carrying out large-scale (5 mL) reactions. All transcriptions proceeded for 3 h at 37 °C. After transcription, the samples were extracted with acid phenol:chloroform, ethanol precipitated, purified by preparative denaturing polyacrylamide gel electrophoresis, and electroeluted. The samples were then dialyzed five times against UltraPure water and folded by heating for 2 min at 95 °C, snap-cooling on ice, and slowly equilibrating to room temperature.