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Vaccinia Capping System

Catalog # Concentration Size List Price Quantity Your Price
M2080S 10000 units/ml 400 units $213.00
$191.70
Catalog # Size List Price Your Price
M2080S 400 units $213.00
$191.70
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A full system for enzymatic capping based on the Vaccinia virus Capping Enzyme (VCE)

  • Adds 7-methylguanylate cap structures (Cap-0) to the 5′ end of RNA generated by in vitro transcription
  • Cap-0 is required for efficient translation of the RNA in eukaryotic systems
  • mRNA Cap 2´-O-Methyltransferase (NEB #M0366) is required to generate Cap-1 structure that reduces cellular innate immune response when the RNA is used in vivo
  • Isolated from a recombinant source
  • Tested for the absence of endonucleases, exonucleases, RNases
  • Getting ready to scale up RNA synthesis? Download our new technical note “Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production” for a generalized set of recommendations for synthesizing high yields of RNA.
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    Avoiding RNase Contamination

Based on the Vaccinia virus Capping Enzyme (VCE), 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.

Vaccinia capping enzyme is composed of two subunits (D1 and D12). The D1 subunit carries three enzymatic activities (RNA triphosphatase, guanylyltransferase and guanine methyltransferase); all necessary for addition of a complete Cap-0 structure, m7Gppp5'N to 5' triphosphate RNA (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).

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 that carries an engineered His-tagged Vaccine capping gene.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Vaccinia Capping System M2080SVIAL -20 1 x 0.04 ml 10,000 units/ml
  Capping Buffer B2080AVIAL -20 1 x 0.1 ml
  S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
  GTP N2080AVIAL -20 1 x 0.05 ml 10 mM
Application Features
Capping mRNA prior to translation assays/in vitro translation
Labeling 5´ end of mRNA

Properties & 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
Supplement with 0.5 mM GTP and 0.1 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



Materials Sold Separately
Notes
  • (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.
  • 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.
  • 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.
  • 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.
  • For transcripts with known structured 5´ ends, the reaction time can be extended to 60 minutes to improve capping efficiency.
  • 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
  • Shuman, S. (1990). J. Biol. Chem. 265, 11960-11966.
  • Furiichi, Y. et al (1977). Nature. 266, 235-239.
  • Lewis, J.D. and Izaurralde, E (1997). Eur. J. Biochem. 247, 461-469.
  • Iizuka, N. et al. (1994). Mol. Cell. Biol. 14, 7322-7330.
  • Guo, P. and Moss, B. (1990). Proc. Natl. Acad. Sci. 87, 4023-4027.
  • Mao, X. and Shuman, S. (1994). J. Biol. Chem. 269, 24472-24479.
  • Grudzien, E. et al. (2004). RNA. 10, 1479.
  • Ramanathan, A. et al. (2016). Nucleic Acids Res. Sep 19; 44(16), 7511–7526. PubMedID: 27317694
FAQs
Tech Tips
  • 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.
Citations
Additional Citations
  • Wulf, Madalee; Buswell, John; Chan, Siuhong; Dai, Nan; Marks, Katherine; Tzertzinis, George; Whipple, Joe; Correa, Ivan; Schildkraut, Ira; (2019) The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping Sci Rep 9 (1), 8594.PubMedID: 31197197, DOI: 10.1038/s41598-019-45083-5
  • Wulf, M.G., Maguire, S., Humbert, P., Dai, N., Bei, Y., Nichols, N.M., Correa, I.R., Jr., Guan, S (2019) Non templated addition and template switching by Moloney murine leukemia virus MMLV based reverse transcriptases co occur and compete with each other J Biol Chem 294(48), 18220-18231..PubMedID: 31640989, DOI: 10.1074/jbc.RA119.010676
Publications
  • Wulf, Madalee; Buswell, John; Chan, Siuhong; Dai, Nan; Marks, Katherine; Tzertzinis, George; Whipple, Joe; Correa, Ivan; Schildkraut, Ira; (2019). The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping Sci Rep. 9 (1), 8594.PubMedID: 31197197, DOI: 10.1038/s41598-019-45083-5
  • Wulf, M.G., Maguire, S., Humbert, P., Dai, N., Bei, Y., Nichols, N.M., Correa, I.R., Jr., Guan, S (2019). Non templated addition and template switching by Moloney murine leukemia virus MMLV based reverse transcriptases co occur and compete with each other J Biol Chem. 294(48), 18220-18231..PubMedID: 31640989, DOI: 10.1074/jbc.RA119.010676
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Certificate of Analysis
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Safety Data Sheets
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