Post-transcriptional capping is when enzymatic modification at the 5’ end of mRNA is performed as a separate step after the in vitro transcription reaction.
Because enzymatic capping is patent-free, inexpensive, and has a high capping efficiency of 80–100% (Grudzien et al., 2004), many scientists use this approach for capping in vitro-transcribed mRNAs.
One of the major capping enzymes, Vaccinia Capping Enzyme (VCE, Cat. # 2460), adds 7-methylguanylate cap structures (m7G or cap-0) to the 5’ end of in vitro-transcribed, uncapped mRNA. All reagents for setting up the capping reaction—10X Capping Buffer, GTP, and the methyl donor, S-adenosylmethionine (SAM)—are included with our VCE. The process takes less than one hour.
Components
Concentration
Size*
Vaccinia Capping Enzyme
10 U/µl
500 U
10X Capping Buffer
10X
100 µl
S-adenosylmethionine (SAM)
32 mM
100 µl
GTP
10 mM
50 µl
*The 500 U size (Cat. # 2460A) supports 50 x 20 µl reactions. The 2,000 U size (Cat. # 2460B) is also available.
mRNA Cap 2'-O-Methyltransferase (2’-O-MTase, Cat. # 2470) then uses SAM as a methyl donor to add a methyl group at the 2’-O position of the first nucleotide (+1) at the 5’ end of the mRNAs, resulting in the cap-1 structure.
Components
Concentration
Size*
mRNA Cap 2'-O-Methyltransferase
50 U/µl
2500 U
10X Capping Buffer
10X
100 µl
S-adenosylmethionine (SAM)
32 mM
100 µl
*The 2500 U size (Cat. # 2470A) supports 50 x 20 µl reactions. The 10,000 U size (Cat. # 2470B) is also available.
Procedure
VCE and 2'-O-MTase: one-step vs. two-step protocol
VCE and 2’-O-MTase work sequentially. That is, mRNA requires the cap-0 structure created by VCE (or ARCA) before it can be a substrate for 2’-O-MTase. This scenario is the 2-step reaction shown in Figure 1. However, for the 1-step reaction, transfer of the methyl group by 2’-O-MTase can occur simultaneously with the VCE’s capping reaction (Figure 1). Details of the 1-step and 2-step protocols are included in the Data Sheet found on the product page.
Figure 1. Post-transcriptional capping procedure. The reactions performed by Vaccinia Capping Enzyme (VCE, Cat. # 2460) and mRNA Cap 2'-O-Methyltransferase (2'-O-MTase, Cat. # 2470) can be a 1-step or 2-step process. A full protocol is available in the Data Sheet.
