Vaccinia Capping Enzyme

Product Information

Vaccinia Capping enzyme is a recombinant protein from Vaccinia virus. Vaccinia Capping Enzyme can add 7-methylguanylate cap structure (Cap 0) to the 5' end of RNA. In eukaryotes, these terminal cap structures are involved in stabilization, transport, translation of mRNAs. Vaccinia Capping enzyme is composed of two subunits (D1R, D12L), and has three enzymatic activities (RNA triphosphatase, guanylyltransferase by the D1R subunit and guanine methyltransferase by the D12L subunit). All necessary for addition of a complete Cap 0 structure, m7Gppp5'N). In vitro, transcripts can be capped in the presence of reaction buffer, GTP, and the methyl donor (SAM).

Source: Recombinant E.coli
Concentration: 10U/μL
Unit Definition: One unit is defined as the amount of enzyme required to incorporate 10pmol of GTP into an 80 nt transcript in 1 hour at 37℃.
Storage Buffer: 20mM Tris-HCl (pH 8.0), 0.1mM EDTA, 100mM NaCl, 1mM DTT, 0.1%(v/v) Triton X-100, 50%(v/v) glycerol.
Companion Product: 10X Capping Buffer, Cat#ON-073, 500mM Tris-HCl, pH 8.0, 50mM KCl, 10mM MgCl₂, 10mM DTT; S-adenosylmethionine (SAM), 32mM, ON-074; 10mM GTP, ON-075.

Key points of operation

1. Do not resuspend the RNA in an EDTA-containing solution.
2. To enhance the stability of RNA in solution, RNase inhibitor is desired, which can be add to a final concertation of 1U/μL at the time of reaction set-up.
3. SAM is unstable at pH 7-8, 37℃ and should be mixed fresh for each reaction series. We recommend determining how many reactions will be performed and diluting an aliquot of the 32mM stock to 2mM.
4. Heating the solution of RNA prior to incubation with Vaccinia capping enzyme, to remove secondary structure on the 5' end of the transcripts. For highly structured 5' ends, increase the RNA heat-denaturation conditions used. For example, 65℃ for 20 min, 75℃ for 10 min, 85℃ for 5 min, etc.
5. Vaccinia Capping Enzyme and 2'-O-methyltransferase work together to produce the Cap 1 structure.
6. Capping reaction incubation time can be increased up to 3 hours at 37℃ for a high capping efficiency for transcripts with known structured 5' ends.

Security Information

Storage Conditions: -20℃
Quality Assurance: Free of Endonuclease, Exonuclease, and RNase activities.
Physical Purity: ＞95% by SDS-PAGE.

Reference

1. Otto J.P. Kyrieleis, Jonathan Chang, Marcos de la Pena, et al. Structure. 22, 452-465(2014).
2. Konarska, M.M. et al., (1984) Cell 38, 731.
3. Kuge, H. et al., (1998) Nucl. Acids Res. 26, 3208.