Figure 1.

Structure, function and in vitro synthesis of vaccine mRNA. (A) mRNA are single stranded nucleic acids composed of an open reading frame (ORF) encoding the gene of interest, flanked by untranslated regions (UTRs) implicated in translation regulation, a cap at the 5’ end consisting of a N7-methylated guanosine residue, important for translation initiation and immune detection, and a poly(A) tail at the 3’ end, participating in the stability of the mRNA, as well as the stabilisation of the translation initiation complex. (B) In vitro synthesis of mRNA is often performed from a linearised plasmid template. The gene of interest is encoded in the plasmid template downstream of a promoter sequence. E. coli are transformed with the plasmid and cultured in liquid medium containing an antibiotic for which the plasmid encodes a resistance gene, thereby allowing the selection of bacteria that express the plasmid. The plasmid is then purified from the culture and digested using restriction enzymes to obtain a linear DNA template. In vitro transcription of mRNA is performed in the presence of the DNA template, an RNA polymerase and nucleotides triphosphates (NTPs). The capping can be performed by directly adding a cap analogue in the IVT reaction mix (1-step reaction), or alternatively by an enzymatic capping reaction after the IVT. If the poly(A) tail is not encoded in the plasmid, an additional step of polyadenylation is required. Created with BioRender.com.