Cap-0 synthesis using Faustovirus Capping Enzyme (FCE) (NEB #M2081)
The protocol below is suitable for capping 50 µg of RNA using 50 units of FCE. The reaction can be scaled up or down as needed to accommodate more or less RNA.
NOTE: Capping efficiency is strongly influenced by 5´ end accessibility of the RNA substrate. For example, 50 units of FCE fully converts 200 µg of some substrates to Cap-0. 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 FCE. 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/early/2022/06/09/rna.079173.122.long
- Combine up to 50 µg of RNA and nuclease-free H2O to a final volume of 38.0 µl.
- (Optional) Heat at 65°C for 5 minutes.
- Place tube on ice.
- Set up the following reaction on ice (in the order specified):
- Incubate at 37°C for 60 minutes.
|
COMPONENTS |
50 µl REACTION |
FINAL CONC. |
|---|---|---|
|
RNA up to 50 µg (from above) |
38 µl |
|
|
10X FCE Capping Buffer |
5 µl |
1X |
|
SAM (2 mM, diluted from 32 mM stock) |
2.5 µl |
0.1 mM |
|
GTP (10 mM) |
2.5 µl |
0.5 mM |
|
FCE (25 U/µl) |
2 µl |
|
|
Total |
50 µl |
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, 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.
