Cellulose-Based Purification of IVT mRNA

Removal of dsRNA contaminants from 100 to 500 μg IVT mRNA was performed using microcentrifuge spin columns (NucleoSpin Filters, Macherey-Nagel, Düren, Germany), cellulose fibers (C6288, Sigma-Aldrich), and a chromatography buffer containing 10 mM HEPES (pH 7.2), 0.1 mM EDTA, 125 mM NaCl, and 16% (v/v) ethanol. The cellulose was first prewashed, by suspending in chromatography buffer at a concentration of 0.2 g cellulose/mL and incubated for 10 min under vigorous shaking. Next, 700 μL cellulose slurry (0.14 g cellulose) was transferred to a microcentrifuge spin column and centrifuged for 60 s at 14,000 × g. The flowthrough was discarded, and 500 μL chromatography buffer was added to the spin column and shaken vigorously for 5 min to resuspend the cellulose within the spin column. After centrifugation for 60 s at 14,000 × g, the flowthrough was discarded and 100–500 μg IVT mRNA in 500 μL chromatography buffer was added to the spin column containing the prewashed cellulose. During the subsequent 30 min, the IVT mRNA and cellulose slurry were shaken vigorously at room temperature to promote resuspension of the cellulose and its association with the dsRNA contaminant. By centrifugation of the spin column for 60 s at 14,000 × g, the cellulose together with the associated dsRNA contaminant was separated from the unbound single-stranded IVT mRNA in the flowthrough. Where indicated, the unbound fraction containing the single-stranded IVT mRNA was directly transferred to a second spin column containing prewashed cellulose, and the 30-min incubation procedure was repeated (2 cycles of purification). Finally, the purified mRNA was recovered from the unbound fraction by adding 0.1 vol 3 M NaOAc (pH 5.5) and 1 vol isopropanol. The precipitated RNA was collected by centrifugation at 14,000 × g and dissolved in nuclease-free water. Sometimes, the unbound fraction contained a tiny amount of particulate cellulose material that was visible after centrifugation as a white pellet. Cellulose particles were avoided using microcentrifuge spin columns containing a 0.2- or 0.45-μm filter (e.g., Ultrafiltration Spin-Columns, 0.45 μm cutoff, Merck, Darmstadt, Germany).

In some experiments, 6-kb linearized plasmid and 1-kb U-containing dsRNA were mixed and subjected to cellulose-based purification. Plasmid DNA and dsRNA were recovered from the unbound and the cellulose-bound fractions, respectively, and loaded to non-denaturing 1% agarose gels containing 0.005% (v/v) GelRed nucleic acid gel stain. GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific) was loaded as a molecular weight marker. To determine the dsRNA-binding capacity of the cellulose, one cycle of cellulose-chromatography of 0.25–25 μg dsRNA using a spin column filled with 0.14 g cellulose was performed. The presence of dsRNA in the unbound fraction indicated the over-saturation of the cellulose.

For FPLC-based cellulose chromatography of 100 mg IVT mRNA, a suspension of 250 g cellulose in 1,400 mL chromatography buffer (0.18 g/mL) was prepared in a beaker by manually stirring to generate homogeneous slurry. The slurry was then transferred to an XK 50/60 chromatography column (GE Healthcare Life Sciences, Freiburg, Germany), and the outlet of the column was opened, allowing the cellulose bed to settle by gravity. The cellulose-filled column was then connected to Äkta avant 25 FPLC system and washed with 780 mL chromatography buffer (1-column volume) at a flow rate of 5 mL/min. Then 100 mL mRNA sample in chromatography buffer (1 mg/mL mRNA final concentration) was loaded to the column, and the chromatography was performed at a flow rate of 5 mL/min. The UV absorbance of the flowthrough was monitored at 260 nm, and the peak fraction corresponding to the eluted IVT mRNA was collected. Later in the run, the chromatography buffer was changed to nuclease-free water to release the cellulose-bound nucleic acid containing the dsRNA contaminants. For further analysis, the nucleic acid from the collected chromatographic peaks was recovered by adding 0.1 vol 3 M NaOAc (pH 5.5) and 1 vol isopropanol, followed by precipitation.