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Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures

Author

Listed:
  • Masahito Inagaki

    (Nagoya University)

  • Naoko Abe

    (Nagoya University)

  • Zhenmin Li

    (Nagoya University)

  • Yuko Nakashima

    (Nagoya University
    Nagoya University)

  • Susit Acharyya

    (Nagoya University)

  • Kazuya Ogawa

    (Nagoya University)

  • Daisuke Kawaguchi

    (Nagoya University)

  • Haruka Hiraoka

    (Nagoya University)

  • Ayaka Banno

    (Nagoya University)

  • Zheyu Meng

    (Nagoya University)

  • Mizuki Tada

    (Nagoya University)

  • Tatsuma Ishida

    (Nagoya University)

  • Pingxue Lyu

    (Nagoya University)

  • Kengo Kokubo

    (Nagoya University)

  • Hirotaka Murase

    (Nagoya University)

  • Fumitaka Hashiya

    (Nagoya University)

  • Yasuaki Kimura

    (Nagoya University)

  • Satoshi Uchida

    (Kyoto Prefectural University of Medicine
    Kawasaki Institute of Industrial Promotion
    Tokyo Medical and Dental University (TMDU))

  • Hiroshi Abe

    (Nagoya University
    Nagoya University
    Japan Science and Technology Agency
    Nagoya University)

Abstract

Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide maximum capping efficiency of around 80–90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.

Suggested Citation

  • Masahito Inagaki & Naoko Abe & Zhenmin Li & Yuko Nakashima & Susit Acharyya & Kazuya Ogawa & Daisuke Kawaguchi & Haruka Hiraoka & Ayaka Banno & Zheyu Meng & Mizuki Tada & Tatsuma Ishida & Pingxue Lyu , 2023. "Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38244-8
    DOI: 10.1038/s41467-023-38244-8
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    References listed on IDEAS

    as
    1. Vladimir Despic & Samie R. Jaffrey, 2023. "mRNA ageing shapes the Cap2 methylome in mammalian mRNA," Nature, Nature, vol. 614(7947), pages 358-366, February.
    2. Ugur Sahin & Petra Oehm & Evelyna Derhovanessian & Robert A. Jabulowsky & Mathias Vormehr & Maike Gold & Daniel Maurus & Doreen Schwarck-Kokarakis & Andreas N. Kuhn & Tana Omokoko & Lena M. Kranz & Mu, 2020. "An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma," Nature, Nature, vol. 585(7823), pages 107-112, September.
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