Biology subjects

Ranaghan, M. J.

Publications and source records attributed to Ranaghan, M. J..

2 recordsLinked to original sources

bioRxiv2026

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 (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.

biochemistry↗

bioRxiv2026

Double-Stranded RNA Profiling with Mass Photometry

Double-stranded RNA (dsRNA) is a potent immunogenic impurity and its detection is a critical quality attribute in characterizing mRNA therapeutics. Standard analytical methods (e.g., sandwich ELISA) are only able to resolve the bulk presence of dsRNA and cannot characterize the different sub-species that may be present within a mRNA sample.. In this study, we use mass photometry (MP) as a single-molecule analytical platform for the simultaneous detection and characterization of dsRNA impurities in mRNA samples. We demonstrate how ionic strength can interfere with the stability of the mAb/dsRNA complex and measure the binding affinity (1 nM) under a set of parameters for reproducible characterization of the complex. We then leverage the J2 antibody to identify antibody/dsRNA complexes that then resolve dsRNA-positive species within an mRNA sample based on discrete molecular weight profiles. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte to enable the use of higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples.

biophysics↗