Purification of modified mRNA via Cellulose Chromatography

The purification of the cellulose was carried out according to a previously described method.^[35 (link)
^] Cellulose purification was used to remove the dsRNA by‐products from unmodified or chemically (Ψ‐, 5mC‐, or Ψ, 5mC) modified IVT mRNA. The cellulose fibers (Sigma–Aldrich, USA) were suspended in chromatography buffer (10 mm HEPES (pH 7.2), 0.1 mm EDTA, 125 mm NaCl, and 16% (v/v) ethanol) to create a cellulose slurry. This slurry was then subjected to centrifugation for 1 min at 14 000 RCF in a spin column, with this process being repeated twice as a prewashing step. The IVT mRNA, which was dissolved in 500 µL chromatography buffer, was placed into the prewashed cellulose fiber spin column for 30 min and then centrifuged at 14 000 RCF within the spin column for 1 min. This procedure effectively separated the dsRNA contaminants from the ssRNA and was repeated twice for thorough purification. Finally, the chromatography buffer was removed from the separated ssRNA via filtration using an Amicon Ultra system (Merck Millipore, USA), and the IVT mRNA dissolving buffer was changed to sterile‐filtered water treated with diethyl pyrocarbonate (DEPC water).