Cap1 capping system HYJ246
The enzymatic capping kit contains vaccinia capping enzyme, mRNA Cap 2´-O-methyltransferase, and other capping components. It can add cap0 or cap1 structures to the 5' end of uncapped mRNA. This structure is identical to the natural cap structure in eukaryotic organisms and can significantly reduce the immunogenicity of exogenous mRNA, enhance its stability, and improve translation efficiency. The vaccinia virus capping enzyme in this kit is derived from recombinant Escherichia coli carrying the vaccinia virus MR. This enzyme consists of two subunits, D1R and D12L, and possesses three enzymatic activities: RNA triphosphatase, guanylyltransferase, and guanine methyltransferase. The D1 subunit performs the functions of RNA triphosphatase and guanylyltransferase, while the D12 subunit functions as a guanine methyltransferase, specifically adding a 7-methylguanylate cap structure (Cap 0) to the 5' end of RNA.
The mRNA Cap 2´-O-methyltransferase is derived from a recombinant Escherichia coli strain carrying the vaccinia virus mRNA Cap 2´-O-methyltransferase gene. This enzyme uses S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (Cap 0), forming the Cap 1 structure. This adds a methyl group to the 2'-O position of the first nucleotide immediately preceding the cap structure at the 5' end of the RNA.
The Cap 1 capping system catalyzes four enzymatic reactions: ①RNA triphosphatase cleaves RNA 5'-triphosphate into diphosphate. ② RNA guanylyltransferase attaches GTP to the 5'-diphosphate of RNA N1. ③ Guanine 7-methyltransferase, using S-adenosylmethionine as a cofactor, catalyzes the methylation of the 7-nitrogen of guanine. ④ mRNA Cap 2'-O-methyltransferase adds a methyl group to the 2'-O position of the first nucleotide immediately preceding the cap structure at the 5' end of Cap 0-RNA.
This kit can cap 50 μg of RNA in a single reaction, allowing for scale-up to produce milligram quantities of RNA. Capping enhances mRNA translation efficiency, significantly improving mRNA expression in transfection and microinjection experiments.
Components
|
Components |
Concentration |
50T |
|
Vaccinia Capping Enzyme |
10 U/μL |
250 μL |
|
mRNA Cap 2´ -O-Methyltransferase |
50 U/μL |
250 μL |
|
SAM |
32 mM |
50 μL |
|
GTP |
100 mM |
50 μL |
|
Recombinant RNase Inhibitor |
40 U/μL |
150 μL |
|
10×Capping Buffer |
/ |
500 μL |
Storage
Store at -25 to -15°C. Valid for 1 years.
Storage buffer
20 mM Tris-HCl (pH 8.0, 25°C), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% glycerol.
Activity Definition
1. Vaccinia Capping Enzyme: One unit (U) of activity is defined as the amount of enzyme required to completely incorporate 10 pmol of GTP into an 80-nt transcript within 1 hour at 37°C.
2. mRNA Cap 2´-O-Methyltransferase: One unit (U) of activity is defined as the amount of enzyme required to methylate 10 pmol of an 80-nt capped RNA transcript within 1 hour at 37°C.