Messenger RNA (mRNA) normally consists of five regions in the strand: 5′ cap, 5′ untranslated region (UTR), coding sequence (which includes a non-coding start codon, coding sequence, and a non-coding stop codon), 3′ UTR, and 3′ poly(A) tail (see Figure 1). The 5′ cap plays a vital role in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against degradation.
There are multiple types of naturally occurring caps for mRNA, most commonly seen as cap 0, cap 1, and cap 2 (see Figure 2). Cap 0 is methylated on the N7 position of the guanosine cap and is typically referred to as m7G cap, or m7Gppp-. Cap 1 is generated by methylating the ribose 2’O group on the initiating nucleotide, while cap 2 is generated by methylating both 2’O groups on the initiating and the second nucleotide. In the cell, the cap 0 structure is essential for efficient translation of the mRNA into protein. Cap 1 and cap 2 have been shown to be important in enhancing translation efficiency and evading the cellular innate immune response.
mRNA can be produced through enzymatic in vitro transcription (IVT) where plasmid DNA templates are transcribed into mRNA with the help of RNA polymerase enzymes. The 5′ cap can be added to the mRNA structure through two different methodologies: enzymatic capping and co-transcriptional capping.
Enzymatic capping is achieved by adding capping enzymes, such as vaccinia enzyme, after the IVT step. It includes an additional step, and the enzymes are very expensive. Enzymatic capping can generate cap 0 or cap 1 structure.
Co-transcriptional capping, as its name suggests, has the capping process occurring simultaneously with the transcription process. It can be achieved by adding capping analogs in the IVT mixture. The widely known cap analogs include anti-reverse cap analog (ARCA) and Cleancap. ARCA will generate cap 0, and Cleancap will generate cap 1 structure. Cleancap, compared with ARCA, is much higher in capping efficiency (50-80% vs. >95%).
Overall, from a process perspective only, Cleancap is the simplest and most efficient way for capping.
Reference:
- https://en.wikipedia.org/wiki/Messenger_RNA
- Chen, Yu, and Deyin Guo. “Molecular mechanisms of coronavirus RNA capping and methylation.” Virologica Sinica 31 (2016): 3-11.