Development of hydrophobic tag purifying monophosphorylated RNA for chemical synthesis of capped mRNA and enzymatic synthesis of circular mRNA

• Published in: Nucleic acids research
• Main Authors: Ototake, Mami, Inagaki, Masahito, Kimura, Seigo, Onda, Kaoru, Tada, Mizuki, Kawaguchi, Daisuke, Murase, Hirotaka, Fukuchi, Kosuke, Gao, Yinuo, Kokubo, Kengo, Acharyya, Susit, Meng, Zheyu, Ishida, Tatsuma, Kawasaki, Tairin, Abe, Naoko, Hashiya, Fumitaka, Kimura, Yasuaki, Abe, Hiroshi
• Format: Journal Article
• Language: English
• Published: England 17.10.2024
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkae847

We developed phosphorylation reagents with a nitrobenzyl hydrophobic tag and used them for 5'-phosphorylation of chemically or transcriptionally synthesized RNA. The capability of hydrophobic tags to synthesize 5'-monophosphorylated RNA was evaluated based on the yield of the desired oligonucleotides, stability of protecting groups during cleavage/deprotection, separation ability in reverse-phase HPLC (RP-HPLC), and deprotection efficiency after RP-HPLC purification. The results showed that a nitrobenzyl derivative with a tert-butyl group at the benzyl position was most suitable for RNA 5'-phosphorylation. Using the developed phosphorylation reagent, we chemically synthesized 5'-phosphorylated RNA and confirmed that it could be purified by RP-HPLC and the following deprotection. In addition, we demonstrated complete chemical synthesis of minimal mRNA by chemical capping of 5'-monophosphorylated RNA. Ribonucleoside 5'-monophosphates with hydrophobic protecting groups have also been developed and used as substrates to transcriptionally synthesize 5'-phosphorylated RNA with >1000 bases. From the mixture of the by-products and the desired RNA, only 5'-monophosphorylated RNA could be effectively isolated by RP-HPLC. Furthermore, monophosphorylated RNA can be converted into circular mRNA via RNA ligase-mediated cyclization. Circular mRNA expression of nanoluciferase in cultured cells and mice. These techniques are important for the production of chemically synthesized mRNA and circular mRNA.