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

  • Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038/s41467-023-38244-8.
Masahito Inagaki  #  1 ,  Naoko Abe  #  1 ,  Zhenmin Li  1 ,  Yuko Nakashima  1  2 ,  Susit Acharyya  1 ,  Kazuya Ogawa  1 ,  Daisuke Kawaguchi  1 ,  Haruka Hiraoka  1 ,  Ayaka Banno  1 ,  Zheyu Meng  1 ,  Mizuki Tada  1 ,  Tatsuma Ishida  1 ,  Pingxue Lyu  1 ,  Kengo Kokubo  1 ,  Hirotaka Murase  1 ,  Fumitaka Hashiya  2 ,  Yasuaki Kimura  1 ,  Satoshi Uchida  3  4  5 ,  Hiroshi Abe  6  7  8  9
Affiliations
  • 1. Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
  • 2. Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
  • 3. Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto, 606-0823, Japan.
  • 4. Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 210-0821, Japan.
  • 5. Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
  • 6. Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. [email protected].
  • 7. Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. [email protected].
  • 8. CREST, Japan Science and Technology Agency, 7, Gobancho, Chiyoda-ku, Tokyo, 102-0076, Japan. [email protected].
  • 9. Institute for Glyco-core Research (iGCORE), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan. [email protected].
  • # Contributed equally.
PMID: 37169757 DOI: 10.1038/s41467-023-38244-8
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.

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