Cap1 capping system

Description:

The enzymatic capping kit contains vaccinia capping enzyme (Vaccinia Capping Enzyme), mRNA Cap 2´-O-Methyltransferase (mRNA Cap2´-O-Methyltransferase) and other capping related components. It can be used without cap. The cap0 or cap1 structure is added to the 5' end of the mRNA of the structure. This structure is completely consistent with the natural cap structure in eukaryotes and can significantly reduce the immunogenicity of foreign mRNA, enhance its stability, and improve translation efficiency. 

The vaccinia virus capping enzyme in this kit is derived from recombinant E. coli carrying vaccinia virus MR. The enzyme is composed of D1R and D12L subunits and has three enzyme activities (RNA triphosphatase activity and guanylyl transferase activity). and guanine methyltransferase activity), the D1 subunit performs the functions of RNA triphosphatase and guanosyl transferase, the D12 subunit performs the function of guanine methyltransferase, and can specifically convert 7-methylguanylate Hat structure (Cap 0) Add to the 5' end of RNA. 

The mRNA Cap2´-O-methyltransferase is derived from a recombinant E. coli strain carrying the vaccinia virus mRNA Cap 2´-O-methyltransferase gene. This enzyme can utilize S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap0), thereby forming the Cap1 structure, i.e., the first cap structure at the 5′ end of the RNA. Add a methyl group to the 2´-O position of each nucleotide.

The Cap1 capping system catalyzes four enzymatic reactions: ① RNA triphosphatase cleaves RNA 5'-triphosphate into diphosphate. ②RNA guanylyltransferase connects GTP to the 5'-diphosphate of RNA N1. ③Guanine 7-methyltransferase uses S-adenosylmethionine as a cofactor to catalyze the 7-nitromethylation of guanine. ④mRNA Cap 2'-O-methyltransferase can add a methyl group to the 2'-O of the first nucleotide of the cap structure at the 5' end of Cap 0-RNA.

Transportation and storage:

Transport at ≤0℃; store at -25℃~-15℃; avoid repeated freezing and thawing.

Unit definition

Vaccinia Capping Enzyme: The amount of enzyme required to completely incorporate 10 pmol GTP into the 80nt transcript within 1 hour at 37°C is defined as 1 activity unit (U). mRNA Cap 2´-O-Methyltransferase: The amount of enzyme required to methylate 10 pmol of 80nt capped RNA transcript within 1 h at 37°C is defined as one activity unit (U).