RNA self-association limits the removal of double-stranded RNA by affinity chromatography

Double-stranded RNAs are inflammatory byproducts of in vitro transcription of single-stranded RNA. Here we investigate removal of dsRNA byproducts using dsRNA affinity chromatography (dsRNA-AC) and two mRNA molecules with contrasting biophysical properties: 1) GFP, and 2) high dsRNA wild-type firefly luciferase (fLucWT). For GFP mRNA, dsRNA reduction was strong, and mRNA recovery was high. In costron, dsRNA removal was inefficient for fLucWT, and mRNA yields were lower. Biophysical characterization revealed that fLucWT contains polydisperse higher-order structures (HOS) that interfered with dsRNA-AC at mRNA concentrations of 0.5 mg/ml. Decreasing fLucWT concentrations restored the effectiveness of dsRNA-AC. dsRNA levels were measured with ELISA, dot-blots, immuno-northern blots, and a cell-based interferon release assay, enabling comparison and cross-validation of these analytical methods. dsRNA-AC is a new chromatography modality, and this study identifies sample polydispersity as a key determinant of efficient dsRNA removal.