Vaccinia Capping System
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
- 2′O Methyltransferase (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
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).
Product SourceAn E. coli strain that carries the genes for the Vaccinia (WR) capping enzyme.
The following reagents are supplied with this product:
Store at (°C) Concentration Capping Buffer -20 10 X S-adenosylmethionine (SAM) -20 32 mM GTP -20 10 mM
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