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

Clark, N. E.; Mallory, H.; Ndong, C. J.; Schraut, M. R.; Winters, R. A.; Freise, J.; Kier, B. L.; Ranaghan, M. J.; Deprez, L.; Dillen, S.; Kearns, K.; Zhu, J.; Scanlon, T. C.

doi:10.64898/2026.09.21.749193

bioRxiv · 10.64898/2026.09.21.749193

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

Clark, N. E.·Mallory, H.·Ndong, C. J.·Schraut, M. R.·Winters, R. A.·Freise, J.·Kier, B. L.·Ranaghan, M. J.·Deprez, L.·Dillen, S.·Kearns, K.·Zhu, J.·Scanlon, T. C.

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Abstract

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 (fLucWT). For GFP mRNA, dsRNA levels decreased by 2,000-fold, and mRNA recovery was high. In contrast, dsRNA levels decreased only two-fold for fLucWT, and mRNA yields were lower. Biophysical characterization revealed that fLucWT 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 fLucWT 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.

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Clark, N. E., Mallory, H., Ndong, C. J., Schraut, M. R., Winters, R. A., Freise, J., Kier, B. L., Ranaghan, M. J., Deprez, L., Dillen, S., Kearns, K., Zhu, J., Scanlon, T. C.. 2026-09-22. RNA self-association limits the removal of double-stranded RNA by affinity chromatography. https://doi.org/10.64898/2026.09.21.749193

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