The mRNA Cap 2´-O-methyltransferase is a recombinant protein derived from vaccinia virus, it can add a methyl group to the 2'-O at the 5' end of the RNA immediately adjacent to the first nucleotide of the cap structure. This enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped Cap 0 to Cap 1. Cap 1 structure can enhance the translation efficiency of mRNA and reduce the immunogenicity of mRNA structure itself, so it helps to improve the expression level of encoded protein after mRNA transfection.
Storage Solution: 50 U/μl mRNA Cap 2'-O-Methyltransferase in 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 50% Glycerol (pH 8.0 @ 25°C)
10* Capping Buffer: 500 mM Tris-HCl, 50mM KCl, 10 mM MgCl2, 10mM DTT (pH 8 @ 25°C)
1. Combine uncapped RNA and nuclease-free water in a final volume of 14.0 μl.
2. Heat at 65°C for 5 minutes.
3. Place tube on ice for 5 minutes.
4. Add the following components in the order specified:
Components | Volume |
Denatured uncapped RNA (from above) | 14.0 μl |
10* Capping Buffer | 2.0 μl |
GTP (10 mM) | 1.0 μl |
SAM (4 mM, dilute 32 mM stock to 4 mM) | 1.0 μl |
Vaccinia Capping Enzyme (10 U/μl) | 1.0 μl |
mRNA Cap 2´-O-Methyltransferase (50 U/μl) | 1.0 μl |
Note: Use of RNase Inhibitor is recommended to enhance stability of RNA in the reaction. Add 0.5 μl of RNase Inhibitor during reaction set up. Subtract the additional volume from the amount of H2O used in the reaction.
5. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).
6. Proceed with purification of the RNA (if required) for downstream applications.
1. The capping reaction efficiency is affected by the structure of the RNA 5' end, so it is recommended to open the advanced structure of the RNA 5' end by thermal denaturation (heating at 65°C for 5 min, placing on ice for 5 min). Denaturation conditions can be adjusted according to the structural complexity of the 5' end of RNA. If the 5' end has no advanced structure, this step can be omitted.
2. The capping reaction can generally be completed within 60 min. If the RNA 5' end structure is complex or the RNA length is short (≤ 200 nt), the reaction time can be extended to 120 min.
3. SAM is unstable at pH 7-8, 37°C and needs to be freshly configured before the reaction starts. To avoid SAM degradation, the working solution needs to be stored on ice.
4. RNase inhibitors can be added to the reaction system to prevent RNase contamination, and the recommended concentration is 1-2 U/μl.
5. The Cap1 capped RNA of this product can add a Poly(A) sequence at the 3' end through Poly(A) polymerase to form a complete mRNA, which can be used for subsequent transfection experiments or in vitro translation experiments.
One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt long capped RNA transcript in 1 hour at 37°C.
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