Product Class: Other

mRNA Cap 2'-O-Methyltransferase
NEBU cloned at NEB recombinant 37 SAM

GMP-grade reagent also available. Learn more.

Product Introduction

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.

Catalog # Size Concentration
M0366S 2000.0 units 50000 units/ml

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Product Information

Description

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

A His-tagged engineered variant of E. coli that carries the gene for Vaccinia mRNA Cap 2´-O-Methyltransferase.
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Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

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.

Reaction Conditions

1X Capping Buffer
Supplement with 0.2 mM S-adenosylmethionine (SAM)
Incubate at 37°C

1X Capping Buffer
50 mM Tris-HCl
5 mM KCl
1 mM MgCl2
1 mM DTT
(pH 8 @ 25°C)

Storage Buffer

20 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% (w/v) Triton® X-100
pH 8 @ 25°C

Application Features

  • 2´-O-methylation of capped in vitro transcripts helps the RNA evade innate immune response in vivo. 

Product Notes

  1. (READ PRIOR TO SETTING UP REACTION)

    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.

  2. 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.

  3. 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

  1. Hyde J.L. and Diamond, M.S. (2015). Virology. 66-74, 479-480.
  2. Devarkar, S.C. et al. Proc Natl Acad Sci U S A.. 113(3), 596-601.
  3. Barbosa, E. and Moss, B. (1978). J. Biol. Chem. 253, 7698-7702.
  4. Lockless, S.W. et al. (1998).  Biochemistry. 37, 8564-8574.

Protocols, Manuals & Usage

Protocols

  1. One-Step Cap-1 mRNA synthesis with Vaccinia Capping Enzyme (VCE) and mRNA Cap 2´-O-methyltransferase (2´-O-me) (NEB #M0366, #M2080)
  2. 2´-O-Methylation of Capped RNA (M0366)

Application Notes

FAQs & Troubleshooting

FAQs

  1. What kind of RNA can be methylated using the mRNA Cap 2'-O-Methyltransferase?
  2. I want to use the mRNA Cap 2'-O-Methyltransferase with the Vaccinia Capping System (NEB# M2080) in a one-step reaction to produce cap 1 RNA. Is that possible?
  3. What precautions should be taken while using the mRNA Cap 2'-O-Methyltransferase?
  4. What is Cap-0 and Cap-1?
  5. Can Vaccinia Capping Enzyme (NEB #M2080) use cap analogs in capping reaction?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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