{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "The University of Sheffield"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 6,
  "authors": [
    {
      "name": "E. P. Welbourne"
    },
    {
      "name": "G. R. Owen"
    },
    {
      "name": "L. P. Wright"
    },
    {
      "name": "S. Trabulo"
    },
    {
      "name": "Z. Kis"
    },
    {
      "affs": [
        0
      ],
      "name": "M. J. Dickman"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/33ewv8p2g0d9h.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.23.753728",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1.full",
  "id": "33ewv8p2g0d9h",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "1607|Mass Spectrometry Techniques and Applications"
    ]
  ],
  "openalex": "W7214152736",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-23",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/33ewv8p2g0d9h.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:15Z",
      "native_id": "10.64898/2026.09.23.753728",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.23.753728v1"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:1607|Mass Spectrometry Techniques and Applications",
      "subfield": "physical"
    }
  ],
  "title": "Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows",
  "title_text": "Multi-attribute characterisation of mRNA via MazF endoribonuclease and LC-MS workflows",
  "type": "preprint",
  "updated_at": "2026-10-30T12:24:15Z",
  "url": "https://research.draconitic.com/paper/33ewv8p2g0d9h",
  "venue": "bioRxiv",
  "versions": []
}