Protocol for one-pot Cap-1 Synthesis with Faustovirus Capping Enzyme (FCE, NEB #M2081) and mRNA Cap 2´-O-methyltransferase (2´-O-me, NEB #M0366)
The protocol below is suitable for capping and 2’-O-methylating 50 µg of RNA using 50 units of FCE and 200 units of mRNA Cap 2’-O-methyltransferase. The reaction can be scaled up or down as needed to accommodate more or less RNA.
NOTE: The efficiencies of both FCE and mRNA Cap 2´-O-me are strongly influenced by 5´ end accessibility of the RNA substrate. For example, 50 units of FCE along with 50 units of mRNA Cap 2’-O-me fully converts 50 µg of some substrates to Cap-1 using the conditions described below. If capping yield for 50 µg of RNA is satisfactory using this recommended protocol, the reaction composition and scaling can be further optimized to cap more RNA while using the same or lower amounts of both FCE and mRNA Cap 2’-O-methyltransferase. We recommend scaling the reaction volume proportionately for larger amounts of RNA (e.g., 100 µl for 100 µg of RNA).
For highly structured substrates, more capping enzyme and longer reaction times may be required to yield complete capping. Additionally, increasing the reaction temperature can also improve capping yields.
A method for measuring capping efficiency is detailed in the following article: https://rnajournal.cshlp.org/content/28/8/1144
- Combine up to 50 µg of RNA and nuclease-Free water to a final volume of 34 µl.
- (Optional) Heat at 65°C for 5 minutes
- Place tube on ice.
- Set up the following reaction on ice (in the order specified):
50 µl REACTION
RNA up to 50 µg (from above)
10X FCE Capping Buffer
4 mM SAM
10 mM GTP
FCE (25 U/µl)
Cap 2'-O-me (50 U/µl)
- Incubate at 37°C for 60 minutes.
RNA is now capped and ready for use in downstream applications. Some applications may require RNA to be purified prior to use. If the RNA needs a poly(A) tail, NEB Poly(A) Polymerase (NEB #M0276) can be used.
NOTE: The optional 65°C heating step is intended to reduced RNA secondary structure prior to capping. This step is not necessary for all substrates and can be omitted.