Cap1 capping system

Cap1 capping system contains Vaccinia Capping Enzyme, mRNA Cap 2'-O-Methyltransferase, and other capping-related components, which can incorporate cap0 or cap1 structures at the 5' end of mRNAs with no cap structure. This structure is identical to the natural cap structure in eukaryotic organisms, which can significantly reduce the immunogenicity of exogenous mRNAs, enhance their stability, and improve translation efficiency. The Vaccinia Capping Enzyme in this kit is derived from recombinant E. coli carrying cowpox virus MR. The enzyme is composed of D1R and D12L double subunits, and has three enzyme activities (RNA triphosphatase activity, Guanylyltransferase activity, and N7-methyltransferase activity). The D1 subunit performs the functions of RNA triphosphatase and Guanylyltransferase. And the D12 subunit performs the function of N7-methyltransferase, specifically adds the 7-methylguanosine cap structure (Cap 0) to the 5' end of RNA. mRNA Cap 2'-O-methyltransferase was derived from a recombinant Escherichia coli strain harboring the cowpox virus mRNA Cap 2'-O-methyltransferase gene. The enzyme can use S-adenosine methylamine (SAM) as a methyl donor to methylate capped RNA (Cap 0), resulting in the formation of a Cap 1 structure, which is the addition of a methyl group at the 2'-O position of the first nucleotide immediately adjacent to the cap structure at the 5' end of the RNA. The Cap1 capping system catalyzes four enzymatic reactions (Figure 1): (i) RNA triphosphatase cleaves RNA 5'-triphosphate to diphosphate. (ii) Guanylyltransferase ligates GTP to the 5'-diphosphate of RNA N1. (iii) N7-methyltransferase catalyzes the 7-nitrogen methylation of guanine using S-adenosylmethionine as a cofactor. (iv) mRNA Cap 2'-O-methyltransferase adds a methyl group to the 2'-O of the first nucleotide immediately adjacent to the cap structure at the 5' end of Cap 0-RNA. This kit can cap 50 μg RNA in a single reaction and can be scaled up to produce milligram-sized RNA. The expression of mRNA in transfection and microinjection experiments can be significantly improved by increasing mRNA translation efficiency after capping.