Nature Communications (May 2023)

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

  • 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,
  • Kengo Kokubo,
  • Hirotaka Murase,
  • Fumitaka Hashiya,
  • Yasuaki Kimura,
  • Satoshi Uchida,
  • Hiroshi Abe

DOI
https://doi.org/10.1038/s41467-023-38244-8
Journal volume & issue
Vol. 14, no. 1
pp. 1 – 17

Abstract

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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.