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’ 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’-ACA” cleavage enables rapid characterisation via oligoribonucleotide mass mapping providing complete sequence coverage and simultaneous assessment of 5-capping efficiency and 3’-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’-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.