Table 1.

The naturally existing caps of mRNAs and the synthesized caps used for mRNA vaccination.

A/The most common natural quanosine methylated mRNA cap modifications and their biological functions
Name of cap modifications and enzymes involved	Structure and use by different species	Site of capping and biological function
Cap O: sequential methylation of the first quanosine nucleotide [5,6] RNA TPase GTase guanine-N7 MTase	m7G(5′)ppp(5′)G Universal for all eukaryotic mRNAs Used by most viruses	Nuclear mRNA capping Recruitment of pre-mRNA protein complex for splicing, polyadenylation and nuclear export. Protection from nonsense mediated decay. Efficient nuclear export by cap binding complex (CBC) Cytoplasm Protection from exonuclease cleavage Affix of elF4E-p to assemble the elF4F complex for initiation of translation. Regulation of gene expression by CBC and elF4E-p.
Cap N6A: substitution of first transcribed guanosine nucleotide by adenosine methylated at 6N position [20,21,22] Multi component protein complex consisting of catalytic subunit Methyltransferase Like 3 (METTL3)	m7G(5′)ppp(5′)AmpNp 20–50% of m7G(5′)ppp(5′)Xm mRNA caps in Hela cells Common to human and mouse mRNA Used by selected viruses including Influenza A virus (IAV), HCV, HBV, HIV, Simian virus 40 (CV40), and enterovirus 71	Co-transcriptional modification. Control of mRNA splicing. Depriving decapping activity. Transcription start site (TSS) signaling. Epitranscriptomic gene regulation. Regulation of viral infection and host immune response. Promotion of RNA Decay. Cap independent mRNA translation.
Cap 1: Methylation of the +1 ribonucleotide at the 2′O position of the ribose [6] m7G-specific 2′O methyltransferase (2′O MTase) cap methyltrasnferase 1 (CMTR1)	m7G(5′)ppp(5′)Gm Lower and higher eukaryotes (mouse and human) Selected viruses	Eukaryotes: nuclear co-transcriptional modification. Restriction of Cap O dependent initiation of translation “as non self” during cellular evasion of foreign mRNA. Promotion of antiviral response by induction of interferon stimulated gene (ISG) proteins. Type 1 interferon signaling leads to expression of interferon-induced proteins with tetratricopeptide repeats (IFIT). Binding of IFIT 1 to Cap O instead of elF4E-p attenuates mRNA translation and replication of viruses that do not encode their own 2′O Mtase, e.g., SARS-CoV, West Nile virus. Viruses: post transcriptional modification at cytoplasm. Evasion of recognition by the innate immune response of the host and interferon response by viruses using a) host mRNA capping pathways (conventional capping) e.g., herpesviruses and retroviruses or (b) viral (non-conventional) e.g., coronaviruses and paramyxoviruses expressing their own 2′O Mtase as in case of SARS-CoV-2.
Cap 2: Methylation of the +2 ribonucleotide at the 2′O position of the ribose [7] CMTR-2	m7G(5′)ppp(5′)GmNm Higher eukaryotes (human)	Nuclear and cytoplasmic Independent from N7 methylation of guanosine (Cap O) or from methylation of first nucleotide 2′O ribose (Cap 1). Greater affiliation for cap2 methylation in Cap 1 mRNAs. Used for efficient pre-mRNA splicing (small nuclear RNAs). Promotion of Cap 1 dependent mRNA restriction of translation as “non self” against foreign (viral) mRNA.
Cap NAD+: 5′ end NAD+ [9] Bacterial RNA polymerase (RNAP) Eukaryotic RNAP II Sensitive to nuclear migration protein nudC (containing NAD-capped RNA hydrolase) Sensitive to DXO degradation proteins in human	NAD(5′)pNp Bacteria Yeasts Human	Nuclear during initiation of transcription, -3- 4-fold increase in mRNA stability in bacteria. Non canonical initiating nucleotide (NCIN)-mediated initiation of transcription. Mammalian cells are equipped with a distinct NAD+ capping mechanism from transcription initiation that does not support cap-dependent translation. Combination of de-NADing proteins in human control the mRNA decay mechanisms and the gene expression.
B/The synthetic mRNA capping systems
Biochemical synthesis pathways	Structure	Biological properties
5′ terminal mRNA modification by vaccinia virus enzymes [14] Guanylyl transferase and S adenosylmethionine: mRNA (guanine-7) methyltransferase	G(5′)ppp(5′)Gp G(5′)ppp(5′)Ap under presence of S adenosylmethionine m7G(5′)ppp(5′)Gmp m7G(5′)ppp(5′)Amp	Post-transcriptional modification No sequence specificity apart from terminal purine. Acting by function of poly(A) as a substrate for all methylation reactions.
Anti-reverse cap analogs (ARCA) [17] Biochemical modifications using pyrophosphate bond formation reactions	7 methyl(3-deoxy) GpppG 7 methyl(3-O-methyl)GpppG 7 methyl(3-O-methyl)GpppG m7G(5′)ppp(5′)G	Inhibit reverse capping of m7GpppGm by bacteriophage polymerases. Increase 2.3-2.6 fold of mRNA translation compared to m7GpppG cap in a rabbit reticulocyte lysate system Increase protein translation in a dose dependant manner in EL4 cancer cells In combination with optimum poly(A) length and β-globin 3′utranslated regions significantly improve RNA stability in immature dendritic cells (human).
Capping enzyme system and 2′O-methyltrasferase to generate Cap 1 [20]	7-methylGpppGm	Contribution to enhancement of transgene expression in stimulated T cells (human) in combination with optimized UTRs and poly(A) lengths. Equivalent in translation efficiency to the ARCA capping system.
Imidodiphosphate: NH analogues, [18] Methylenebisphosphonate:CH2 analogues, [18] Dihalogenmethylenebisphosphonate: CCl2 and CF2 analogues, [18] Biochemical moiety substitution within the 5’,5’-tri- or tetraphosphate bridge of mRNA caps (including ARCAs)	…5′pNHp5′… …5′pCH2p5′… …5′pCl2p5′… …5′pCF2p5′…	Sequential increase in binding affinity to the elF4E Decrease degradation by DcpS (decapping scavenger in exosome) and decapping complex Dcp1–Dcp2
Cap analogues with 1,2-dithiodiphosphate moieties [19] Biochemical dithiodiphosphate modified nucleotide synthesis applied to ARCA synthesis 2S analogs: phosphorothioate (O-to-S) substitution inside the triphosphate bridge 2S ARCA analogs: modified by the presence of a 2′-O-methyl group	7,2′-O-dimethylGppSpGD1 7,2′-O-dimethylGppspsGD1D2 7,2′-O-dimethylGpppspsGD1D2	Dramatic increase in ElF4E-p binding affinity Decrease of decapping susceptibility by SpDcp1/2 enzyme complex Increasing efficiency of mRNA translation in immature dendritic cells (D1 to D1D2) D1: gold standard for the treatment of melanoma