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Updated: Jan 18, 2026

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
Biophysical characterisation of mRNA structure and its impact on integrity analysis by liquid chromatography and
Mayada Elgohary1, Alexandra L J Webb2, Joseph C Ward3
1Analytical Sciences, BioPharmaceuticals Development, BioPharmaceuticals R&D, AstraZeneca, Cambridge Biomedical Campus, Cambridge CB2 0AA, United Kingdom.
Abstract:
Messenger RNA (mRNA) integrity is a critical quality attribute (CQA) commonly assessed using liquid chromatography (LC) and capillary gel electrophoresis (CGE). The LC and CGE methods used for integrity tests are also used for stability studies for establishing product shelf-life for mRNA-based therapeutics. Advances in sequence and structure engineering have demonstrated significant improvements in mRNA translatability and stability. However, the higher order structure (HOS) of mRNA remains poorly understood and it is difficult to correlate experimentally measured structures with their bioinformatically predicted conformations. Previous studies have shown that mRNA HOS can cause artefactual bands or peaks in CGE and LC analyses, leading to anomalous integrity readouts. In this study, we employed a range of biophysical methods to characterise mRNA HOS, aiming to clarify the origins of the additional peaks observed in CGE and LC analyses and to better comprehend the influences of predicted minimum free energy (MFE) and structural features on mRNA integrity, translatability, and stability. Our findings provide new method-related considerations for measuring mRNA integrity as well as insights into the effects of mRNA structure on stability under long term storage and forced degradation conditions.
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