Biology subjects

Owen, G. R.

Publications and source records attributed to Owen, G. R..

3 recordsLinked to original sources

bioRxiv2026

Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows

The rapid expansion of mRNA-based medicines has driven demand for robust, high-resolution analytical methods capable of characterising critical quality attributes including sequence identity, 5[prime] capping efficiency, and poly(A) tail length and heterogeneity. Here, we present two complementary liquid chromatography-mass spectrometry based workflows for mRNA characterisation, both based on site-specific digestion with the endoribonuclease MazF from Escherichia coli. A middle-up approach, optimised via specific 5[prime]-ACA cleavage enables rapid characterisation via oligoribonucleotide mass mapping providing complete sequence coverage and simultaneous assessment of 5[prime]-capping efficiency and 3[prime]-poly(A) tail length and heterogeneity. This workflow was successfully applied to three different mRNA constructs, NLuc, eGFP and FLuc mRNAs, achieving sequence coverages of 100%, 87%, and 74%, respectively. A complementary bottom-up approach, employing less specific 5[prime]-ACX cleavage enables tandem mass spectrometry-based sequencing of shorter oligoribonucleotides and detailed characterisation via mRNA sequence mapping. Applied to SARS-CoV-2 Spike Protein mRNA, this method yielded 50% sequence coverage based on unique oligoribonucleotides with MazF alone, extended to 90% by combining MazF, partial RNase T1, and partial RNase U2 digests. Together, these workflows provide a flexible, orthogonal platform for both high-throughput quality assessment and in-depth primary sequence characterisation of mRNA vaccines and therapeutics.

biochemistry↗

bioRxiv2026

DNA template heterogeneity and in vitro transcription reaction conditions impact the poly(A) tail length and heterogeneity of mRNA

mRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

biochemistry↗

bioRxiv2026

Loss of α2,3-linked sialoside in the receptor-binding site of a H5N1 influenza hemagglutinin identified in a human patient

In November 2024, an adolescent female in British Columbia was hospitalized presenting with severe symptoms including respiratory failure due to infection with a novel H5N1 subtype influenza strain (BC24). Using cryogenic electron microscopy (cryo-EM), we show here that the N169 2,3-linked auto-glycan that is found in the sialic acid binding site of previously studied H5 hemagglutinin (HA) proteins is absent in purified BC24 HA protein, suggesting greatly reduced affinity for 2,3-linked sialosides. Glycan microarray analysis shows that the BC24 HA protein displays reduced or no binding not just to most 2,3-linked sialosides, but also to 2,6-linked sialosides. Full-length BC24 HA expressed in A549 lung alveolar carcinoma cells drives membrane fusion, albeit at significantly lower levels than previous H5 HA proteins, and post-infection sera from the patient display strong binding to BC24 HA and HA proteins from other influenza subtypes. The high virulence of the BC24 strain despite weak receptor binding reveals further complexity in the factors that result in severe disease caused by avian influenza.

biochemistry↗