A new hydrophobic tag enables the high-purity production of a key intermediate in the chemical synthesis of capped mRNA and circular RNA. These RNAs are used in research and therapeutics such as mRNA vaccines and are normally expensive to make because of the way they are produced.
RNA for therapeutic purposes is typically synthesised enzymatically via in vitro transcription or, more recently, using chemical synthesis. The latter allows for the introduction of precise chemical modifications to enhance translational activity, is more easily scalable and is faster than enzymatic methods. ‘In order to produce mRNA through transcription, it would take more than a month to synthesise the template DNA,’ explains Hiroshi Abe of Nagoya University, Japan, who led the work. ‘On the other hand, the chemical synthesis method for mRNA can significantly reduce production time by skipping the synthesis of the DNA template.’ Both RNA synthesis methods produce 5’-monophosphate, the in demand intermediate to make mRNA, but also 5’-triphosphate RNA that is difficult to separate out and impedes further reactions.
The team developed a phosphorylation reagent bearing a hydrophobic tag to simplify the RNA purification process. After screening several candidates, the team determined that a nitrobenzyl derivative with a tert-butyl group at the benzyl position changed the chemical properties of the RNA to allow for simpler separation of 5’-monophosphorylated RNA from 5’-triphosphorylated species. The tag can then be removed by brief exposure to UV light, or through reductive conditions. Simplifying this separation process removes one significant barrier still holding back chemical synthesis of mRNA.