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.
This product is related to the following categories:
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
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.
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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