Fig. 5. mRNA capping procedure using capping enzymes or cap analogs.

A Production of post-transcriptional modifications of mRNA with cap0 requires three enzymes: triphosphatase, guanylyltransferase, and N7-methyltransferase with S-adenosylmethionine (SAM) as the methyl donor. Subsequently, the cap0 is modified with 2′-O-ribose methyltransferase to generate the cap1 structure. B Cap analogs commonly used for in vitro transcription of mRNA are CleanCap® Reagent AG (TriLink) and CleanCap® Reagent AU (TriLink). The proposed mechanism of CleanCap co-transcriptional initiation involves the docking of AmG or AmU dimers onto the +1 and +2 positions in template nucleotides. Initiation occurs upon coupling of CleanCap with an nucleoside triphosphate (NTP) occupying the +3 position.¹⁵⁷