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 common mRNA molecules: 1) GFP, and 2) high dsRNA wild-type firefly luciferase (fLuc _WT ). For GFP mRNA, dsRNA levels decreased by 2,000-fold, and mRNA recovery was high. In contrast, dsRNA levels decreased only two-fold for fLuc _WT , and mRNA yields were lower. Biophysical characterization revealed that fLuc _WT contains polydisperse higher-order structures that interfered with dsRNA-AC. Sedimentation-velocity analytical ultracentrifugation experiments demonstrated the higher-order structures were driven by mRNA concentration. Decreasing fLuc _WT concentrations increased the effectiveness of dsRNA-AC by reducing polydispersity. 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.