{
  "abstract": "In addition to the 5' Cap-1 structure, the initiating adenosine of mammalian mRNA can be further modified by PCIF1 (Phosphorylated C-terminal domain-interacting factor 1) to form 2'-O,N6-dimethyladenosine (m6A Cap-1). While the biological role is still under investigation, the implications of m6A Cap-1 in mRNA metabolism and viral-host interactions have spurred interest in PCIF1 and m6A Cap-1 as biomarkers, drug targets, and research tools. Here, we identify and characterize two PCIF1-like cap-specific N6-adenosine methyltransferases, PC04 and PC05 and evaluate their applicability in m6A Cap-1 mRNA synthesis in vitro. We found that both enzymes are highly specific to the m7G cap structure and highly active on Cap-1 RNA oligonucleotides and trinucleotide Cap-1 analogs at nanomolar concentrations. We demonstrate that PC04 or PC05 methylation reactions can be incorporated into established post-transcriptional capping and cap analog-based co-transcriptional in vitro transcription (IVT) workflows to produce m6A Cap-1 RNAs. Finally, we show that PC05 can be combined with a previously reported FCE::T7 RNA polymerase fusion to synthesize m6A Cap-1 mRNA from DNA templates in a single step, co-transcriptional manner. The robustness of the PCIF1-like cap N6-adenosine methyltransferases can accelerate research into the biological roles of cap m6A modification and development of highly effective mRNA-based therapeutics.",
  "abstract_shown": true,
  "affiliations": [
    "New England Biolabs, Inc."
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "name": "D. W. Kneller"
    },
    {
      "name": "D. Nan"
    },
    {
      "affs": [
        0
      ],
      "name": "S. H. Chan"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/3840bjnsb2b2y.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.22.757692",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.22.757692v1.full",
  "id": "3840bjnsb2b2y",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.22.757692v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "1312|RNA modifications and cancer"
    ]
  ],
  "openalex": "W7223384481",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.22.757692v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-22",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/3840bjnsb2b2y.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:29:18Z",
      "native_id": "10.64898/2026.10.22.757692",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.22.757692v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|RNA modifications and cancer",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Cap-specific N6-adenosine methyltransferases and applications in m6A Cap-1 mRNA synthesis",
  "title_text": "Cap-specific N6-adenosine methyltransferases and applications in m6A Cap-1 mRNA synthesis",
  "type": "preprint",
  "updated_at": "2026-10-30T12:29:18Z",
  "url": "https://research.draconitic.com/paper/3840bjnsb2b2y",
  "venue": "bioRxiv",
  "versions": []
}