The phosphorylation reagent with a nitrobenzyl hydrophobic tag allows production of high purity fully chemically synthesized mRNA and transcriptionally synthesized cyclic mRNA. Credit: Reiko Matsushita

In an era where viral outbreaks can escalate into global pandemics with alarming speed, the ability to quickly develop new vaccines has become crucial. However, the speed of vaccine production is limited because the mRNA used in it is partly chemically synthesized and partly synthesized using enzymes, a relatively slow process.

A team of researchers from Nagoya University in Japan has successfully developed an innovative synthesis technology capable of producing high purity, fully chemically-synthesized mRNA, cutting out the slower enzyme reactions.

This advancement establishes a foundation for more rapid reactions to viral outbreaks and emerging diseases, which will hopefully lead to mitigation of future infections at a preliminary stage. Their results were published in the journal Nucleic Acids Research.

Given its significant role in combating the COVID-19 pandemic, mRNA is now widely recognized for its potential to help prevent infectious diseases. Experts anticipate that in the future mRNA technology will be used to treat genetic disorders and emerging illnesses. However, producing mRNA remains challenging because of concerns about purity and production speed.

These problems can be addressed using fully chemically-synthesized mRNA. According to Masahito Inagaki, “One of the most significant advantages of fully chemically-synthesized mRNA is its ability to bypass the complex and time-consuming enzymatic reactions typically required in mRNA production. A method that relies purely on chemical reactions would significantly shorten the production process.”

It also offers benefits to people that have strong immune responses to vaccines. mRNA that is derived from 5′-monophosphorylated RNA is susceptible to contamination by incomplete RNA fragments, causing a strong immune reaction. This immune response increases the risk of side effects, particularly inflammation. However, existing purification technologies have struggled to remove these impurities, limiting its potential.

To address these issues, Professor Hiroshi Abe, doctoral student Mami Ototake, and Assistant Professor Inagaki devised a novel phosphorylation reagent with a nitrobenzyl group that serves as a hydrophobic purification tag.

Inagaki explained, “Nitrobenzyl groups have high hydrophobicity; therefore, when the nitrobenzyl group is introduced into the RNA molecule, the mRNA becomes more hydrophobic. As impure RNA lacks nitrobenzyl groups, it can be easily separated from the target RNA containing nitrobenzyl groups using reverse-phase high-performance liquid chromatography.

“This approach yields pure RNA, free from length inconsistencies and impurities typically associated with transcription-based synthesis methods.”

Besides fully synthesizing mRNA chemically, the team also created pure circular mRNA using the same method. Circular mRNAs are unique because they lack terminal structures, making them resistant to degradation by nucleic acid-degrading enzymes in the body, resulting in a longer-lasting medicinal effect.

The breakthrough in mRNA production has significant implications for the future of medical treatments. “This innovation paves the way for the highly efficient production of fully chemically synthesized mRNA and circular mRNA, which hold the potential to revolutionize RNA drug discovery and expand the scope of mRNA-based treatments,” Abe said.

Faster and purer vaccine production should improve our response times to future infectious threats. In the future, the team hopes to also use these results to develop new mRNA vaccines for cancer antigens and genetic diseases.