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 and Hiroshi Abe ()
Additional contact information
Masahito Inagaki: Nagoya University
Naoko Abe: Nagoya University
Zhenmin Li: Nagoya University
Yuko Nakashima: 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
Hiroshi Abe: Nagoya University

Nature Communications, 2023, vol. 14, issue 1, 1-17

Abstract: 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.

Date: 2023
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-38244-8

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DOI: 10.1038/s41467-023-38244-8

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