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  • Vaccinia Capping System

    Description

    Based on the Vaccinia virus Capping Enzyme, the Vaccinia Capping System provides the necessary components to add 7-methylguanylate cap structures (Cap 0) to the 5´end of RNA (1). In eukaryotes, these terminal cap structures are involved in stabilization (2), transport (3), and translation (4) of mRNAs. Enzymatic production of capped RNA is an easy way to improve the stability and translational competence of RNA used for in vitro translation, transfection, and microinjection. Alternatively, use of labeled GTP in a reaction provides a convenient way to label any RNA containing a 5´ terminal triphosphate.

    This single enzyme is composed of two subunits (D1 and D12) and has three enzymatic activities (RNA triphosphatase and guanylyltransferase by the D1 subunit and guanine methyltransferase by the D12 subunit); all necessary for addition of a complete Cap 0 structure, m7Gppp5´N (5,6). In vitro transcripts can be capped in less than one hour in the presence of the capping enzyme, reaction buffer, GTP, and the methyl donor, SAM. Capping is nearly 100% efficient and all capped structures are added in the proper orientation, unlike co-transcriptional addition of some cap analogs (7).

    Product Source

    An E. coli strain that carries the genes for the Vaccinia (WR) capping enzyme.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    10X Capping Buffer10X
    S-adenosylmethionine (SAM)-2032 mM
    GTP10 mM

    Advantages and Features

    Applications

    Capping mRNA prior to translation assays/in vitro translation
    Labeling 5´ end of mRNA

    Properties and Usage

    Unit Definition

    One unit of Vaccinia Capping Enzyme is defined as the amount of enzyme required to incorporate 10 pmol of (α32P) GTP into an 80 nt transcript in 1 hour at 37°C.

    Reaction Conditions

    1X Capping Buffer
    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 Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 8.0 @ 25°C

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. (read prior to setting up reaction)

      RNA used for capping reactions should be purified prior to use and suspended in nuclease-free water. EDTA should not be present and the solution should be free of salts.
    2. While RNase Inhibitor is not required, many users prefer to use it to enhance the stability of their RNA in solution. If this is desired, 0.5 µl of a standard RNase inhibitor prep (such as RNase Inhibitor, Murine, NEB #M0314) can be added at the time of reaction set-up. The additional volume can be subtracted from the amount of H2O used in step 1 of both the capping and the labeling protocols.
    3. Heating the solution of RNA prior to incubation with the Vaccinia Capping Enzyme removes secondary structure on the 5´ end of the transcript. Extend time to 10 minutes for transcripts with known highly structured 5´ ends.
    4. 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 2 mM just prior to setting up the reactions. This "working stock" should be kept on ice to prevent degradation of SAM.
    5. For transcripts with known structured 5´ ends, the reaction time can be extended to 60 minutes to improve capping efficiency.
    6. For labeling the 5´ end, the total GTP concentration should be around 1–3X the molar concentration of mRNA in the reaction. The 10 mM stock can be diluted and a "spike" of hot GTP added to make the GTP mix.

    References

    1. Shuman, S. (1990). J. Biol. Chem. 265, 11960-11966.
    2. Furiichi, Y. et al (1977). Nature. 266, 235-239.
    3. Lewis, J.D. and Izaurralde, E (1997). Eur. J. Biochem. 247, 461-469.
    4. Iizuka, N. et al. (1994). Mol. Cell. Biol. 14, 7322-7330.
    5. Guo, P. and Moss, B. (1990). Proc. Natl. Acad. Sci. 87, 4023-4027.
    6. Mao, X. and Shuman, S. (1994). J. Biol. Chem. 269, 24472-24479.
    7. Grudzien, E. et al. (2004). RNA. 10, 1479.

    Supporting Documents

    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]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Labeling Protocol (M2080)
    2. Capping Protocol (M2080)
    It is important to ensure that the RNA is purified prior to use. It should be suspended in nuclease-free water free from EDTA and salts.
    Heating the RNA at 65°C for 5 minutes prior to setting up the reaction removes secondary structures on the 5´ end of the transcript. Extend time to 10 minutes for RNA with known highly structured 5’ ends.
    SAM is unstable at pH 7–8, 37°C. Hence, it should be diluted just prior to starting the reaction.
    We strongly recommend wearing gloves, using nuclease-free tubes and reagents, and thoroughly cleaning pipettes and bench surfaces to avoid RNase contamination.