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:10.1038/s41467-023-38244-8

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.

Authors

Masahito Inagaki^#¹, Naoko Abe^#¹, Zhenmin Li¹, Yuko Nakashima^{1

2}, Susit Acharyya¹, Kazuya Ogawa¹, Daisuke Kawaguchi¹, Haruka Hiraoka¹, Ayaka Banno¹, Zheyu Meng¹, Mizuki Tada¹, Tatsuma Ishida¹, Pingxue Lyu¹, Kengo Kokubo¹, Hirotaka Murase¹, Fumitaka Hashiya², Yasuaki Kimura¹, 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. h-abe@chem.nagoya-u.ac.jp.
⁷ Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan. h-abe@chem.nagoya-u.ac.jp.
⁸ CREST, Japan Science and Technology Agency, 7, Gobancho, Chiyoda-ku, Tokyo, 102-0076, Japan. h-abe@chem.nagoya-u.ac.jp.
⁹ Institute for Glyco-core Research (iGCORE), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan. h-abe@chem.nagoya-u.ac.jp.

^# Contributed equally.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cells, Cultured
Protein Biosynthesis*
RNA Caps* / metabolism
RNA, Messenger / genetics
RNA, Messenger / metabolism

Substances

RNA, Messenger
RNA Caps