mRNA Cap 2´-O-Methyltransferase 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.
mRNA cap-1 structure may improve expression during microinjection and transfection experiments
Isolated from a recombinant source
Tested for the absence of endonucleases, exonucleases, RNases
mRNA Cap 2´-O-Methyltransferase 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.
Recently, it has been reported that the 2′-O-methylation in the cap-1 structure helps the mRNA evade innate immune response in some cell types in vivo (1,2).
mRNA Cap 2´-O-Methyltransferase specifically requires RNA with an m7GpppN cap as substrate. It cannot utilize RNA with pN, ppN, pppN or GpppN at the 5´ end (3,4). Capped RNA may be prepared via in vitro transcription using cap analog or through enzymatic capping using the Vaccinia Capping Enzyme (NEB #M2080 ).
The reagents provided in this pack can be used to methylate up to 400 μg of capped RNA.
Figure 1. 5´ Cap Structure
Schematic representation of mRNA 5´ cap structure indicating the 7-methylguanosine, shown in yellow, and the 5´ end of the mRNA, shown in blue. The 2´-O-methyl group present in Cap-1 and Cap-2 structures is shown in red.
Product Source
An E. coli strain carrying a gene encoding for his-tagged variant of Vaccinia mRNA Cap 2´-O-Methyltransferase.
This product is related to the following categories:
RNA prepared using in vitro transcription and cap analog should be purified prior to use and resuspended in nuclease-free water. EDTA and salts should not be present in the solution.
mRNA Cap 2´-O-Methyltransferase may be directly added to a Vaccinia Capping System (NEB #M2080) reaction. RNA purification is not required in this case.
Heating the RNA at 65°C for 5 minutes prior to incubation with the enzyme removes secondary structure on the 5´ end of the transcript. Extend time to 10 minutes for transcripts with known highly structured 5´ ends.
SAM is unstable at pH 7–8, 37°C and should be mixed fresh prior to starting the reaction. We recommend determining how many reactions will be performed and diluting an aliquot of the 32 mM stock to 4 mM immediately before setting up the reactions. This "working stock" should be kept on ice to prevent degradation of SAM.
References
Hyde J.L. and Diamond, M.S. (2015). Virology. 66-74, 479-480. PubMedID: 25682435
Devarkar, S.C. et al. (2016). Proc Natl Acad Sci U S A. 113(3), 596-601. PubMedID: 26733676
Barbosa, E. and Moss, B. (1978). J. Biol. Chem. 253, 7698-7702. PubMedID: 701282,
Lockless, S.W. et al. (1998). Biochemistry. 37, 8564-8574.
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