mRNA Cap-2′-O-Methyltransferase with Buffer

mRNA Cap 2´ -O-methyltransferase was derived from a recombinant E. coli strain that carries the gene for the vaccinia mRNA Cap 2´-O-Methyltransferase. This enzyme adds a methyl group at the 2´-O position of the first nucleotide adjacent to the cap structure at the 5´ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (cap-0) resulting in a cap-1 structure.

The Cap 1 structure can increase the translation efficiency, improving the expression of mRNA in transfection and microinjection experiments. This enzyme specifically requires RNA with an m7GpppN cap as substrate. It cannot utilize RNA with pN, ppN, pppN or GpppN at the 5´ end. Capped RNA may be prepared via in vitro transcription using cap analog or through enzymatic capping using the Vaccinia Capping Enzyme.

Storage -20℃ for storage（Avoid repeated freeze-thaw cycles）

Storage buffer 20 mM Tris-HC（lpH 8.0，25℃), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100， 50% glycerol.

Unit Definition One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt capped RNA transcript in 1 hour at 37°C.

Quality Control Assays

Exonuclease: 50 U of mRNA Cap 2´-O-Methyltransferase with 1 μg λ-Hind III digest DNA at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Endonuclease: 50 U of mRNA Cap 2´-O-Methyltransferase with 1 μg λDNA at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

Nickase: 50U of mRNA Cap 2´-O-Methyltransferase with 1 μg pBR322 at 37 ℃ for 16 hours yields no degradation as determined by agarose gel electrophoresis.

RNase: 50 U of mRNA Cap 2´-O-Methyltransferase with 1.6 μg MS2 RNA for 4 hours at 37 ℃ yields no degradation as determined by agarose gel electrophoresis E.coli DNA: 50 U of mRNA Cap 2´-O-Methyltransferase is screened for the presence of E. coli genomic DNA using TaqMan qPCR with primers specific for the E. coli 16S rRNA locus. The E. coli genomic DNA contamination is ≤0.1 pg/50 U.

Bacterial Endotoxin: LAL-test, according to Chinese Pharmacopoeia IV 2020 edition, gel limit test method, the general rule (1143). Bacterial endotoxin content should be ≤10 EU/mg.

Reaction system and conditions

1. Combine an appropriate amount of Capped RNA and RNase-free H2O in a 1.5 mL microfuge tube to a final volume of 16.0 µL.

2. Heat at 65℃ for 5 minutes followed by an ice bath for 5 minutes.

3. Add the following components in the order specified (for methylation of Capped RNA less than 10 μg):

*10× Capping Reaction Buffer ： 500 mM Tris-HCl(pH 8.0，25℃), 50 mM KCl, 10 mM MgCl2 、10 mM DTT.

4. Incubate at 37℃ for 1 hour (2 hours of incubation is recommended for the target fragment less than 200 nt).

Applications

To improve mRNA expression during microinjection and transfection experiments.

Notes on use

1. Before reaction, RNA should be purified and dissolved in nuclease-free water, all the solutions should not contain any EDTA and ions.

2. It is recommended to heat the sample RNA at 65℃ for 5 min before the reaction to remove the secondary structure on the 5´end of the transcript. It could be extended to 10 min for complex 5´ -terminal structure.
3. For your safety, please wear lab coat and disposable gloves during operation.
4. SAM reagent represents poor stability at pH 7 – 8, 37°C and it should be prepared just before use. The required amount of SAM can be calculated before diluting the 32 mM SAM reserve solution to 4 mM working solution before the reaction. The working solution should be stored on ice in order to prevent degradation.