Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows
- 1The University of Sheffield
Abstract
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.
Related
- Multi-techniques characterisation of anti-reflective Ta2O5 and TaOxNy thin films deposited by reactive sputtering: coupling X-ray photoelectron spectroscopy, scanning/transmission electron microscopy and ion beam analysis
- Experimental Characterisation and Finite Element Modelling of Polyamide-11 Fabricated via Multi Jet Fusion
- Twelve quick tips for designing AI-driven HPC workflows
- Integrating artificial intelligence and molecular simulation for peptide discovery: limitations, validation and decision-centered workflows
- Comparative O-Glycosylation Analysis of Etanercept Originator and Biosimilars Using Orthogonal LC-MS Workflows
BibTeX
@misc{welbourne2026multiattribute,
title = {{Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows}},
author = {E. P. Welbourne and G. R. Owen and L. P. Wright and S. Trabulo and Z. Kis and M. J. Dickman},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753728},
url = {https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows
AU - E. P. Welbourne
AU - G. R. Owen
AU - L. P. Wright
AU - S. Trabulo
AU - Z. Kis
AU - M. J. Dickman
PY - 2026
DA - 2026/09/23
PB - bioRxiv
DO - 10.64898/2026.09.23.753728
UR - https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1
AB - 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.
ER -