Faustovirus Capping Enzyme
NEBU 37 70 Heat SAM

GMP-grade coming soon.

  • Faustovirus Capping Enzyme adds the m7G-cap (Cap-0) to the 5′ end of triphosphorylated and diphosphorylated RNA.
  • Experience improved capping efficiency, even on difficult substrates
  • Achieve robust capping with less enzyme
  • Set up reactions under a broad temperature range, for added flexibility
  • Choose as an alternative to Vaccinia Capping System, with minimal optimization required
  • Enable one-pot Cap-1 synthesis, as FCE is compatible with mRNA Cap 2′-O-Methyltransferase
  • Benefit from no licensing fees from NEB for the use of FCE
 
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Catalog # Concentration Size
M2081S 25,000 units/ml 500 units
M2081L 25,000 units/ml 2,500 units
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  • Product Information
    Faustovirus Capping Enzyme (FCE) catalyzes the addition of N7-methylguanosine cap (m7G) to the 5′ end of triphosphorylated and diphosphorylated transcripts, producing Cap-0 RNA(1). FCE is a single-subunit enzyme that combines the three activities necessary to produce the Cap-0 structure- triphosphatase, guanylyltransferase, and (guanine-N7)-methyltransferase. Installation of Cap-0 is a key step in eukaryotic mRNA maturation along with 2′-O-methylation at position 1 (Cap-1) and polyadenosine (poly(A)) tailing. The Cap-0 structure also promotes RNA stability(2) and prevents inadvertent activation of innate immune responses triggered by triphosphorylated RNA(3)

    FCE retains significant capping activity at low temperatures and tolerates reaction temperatures up to 55°C. In many cases, 1 µl of FCE (25 units) can cap over 100 µg of RNA in 1 hour at 37°C. GTP and S-adenosylmethionine (SAM) are required for capping activity and are included with the enzyme.

    Source: An E. coli strain that carries a plasmid encoding the Faustovirus Capping Enzyme with a C-terminal His-tag.

    Figure 1: FCE offers increased capping efficiency and workflow optimization

    Figure 1

    A. mRNA capping by FCE and Vaccinia Capping Enzyme (VCE) at 37°C. 200 μg (~350 picomoles, 7 μM) of a 1.77 kb FLuc transcript having 5´-UTR sequences as indicated were treated with a limiting amount of FCE (25 units, 1 picomole, 20 nM in 50 μl) or VCE (10 units, 1 picomole, 20 nM in 50 μl) for 1 hour at 37°C. Note that this is less than our recommended amount of enzyme highlighting the increased capping efficiency of FCE vs VCE and the potential benefits of workflow optimization. 

    B. mRNA capping by FCE at 37°C and 42°C. 200 μg (~350 picomoles, 7 μM) of a 1.77 kb FLuc transcript having 5′-UTR sequences as indicated were treated with a limiting amount (25 units, 1 picomole or 20 nM) of FCE for 1 hour at 37°C or 42°C. Note that this is less than our recommended amount of enzyme highlighting the potential benefits of workflow optimization. All capping reactions were performed in 50 μl reactions containing 0.1 mM SAM, and 0.5 mM GTP, 1X FCE Capping Buffer for FCE reactions or 1X Capping Buffer for VCE reactions. Following capping reactions, mRNA capping was measured using targeted RNase H cleavage and LC-MS. 



    Figure 2: FCE expands temperature range for RNA capping reactions

    Figure 2

    RNA capping by FCE and VCE from 15ºC to 60ºC. Capping reactions (20 μl) contained 2.5 μM RNA substrate (5´-triphosphate 20mer 3´-FAM), a limiting concentration (8 nM) of capping enzyme, 0.1 mM SAM, and 0.5 mM GTP. FCE capping reactions were performed in 1X FCE Capping Buffer, and VCE capping reactions were performed in 1X Capping Buffer. All reactions were incubated for 30 minutes at the indicated reaction temperature. Reaction products were measured by capillary electrophoresis. 



    Figure 3. FCE offers robust capping of a variety of RNA 5´ ends (at least 100 μg yield per 50 units)

    Figure 3 

    Extent of mRNA capping by FCE. 200 μg (~350 picomoles, 3.5 uM) of a 1.77 kb FLuc transcript having 5´-UTR sequences as indicated were treated with 2 μl (50 units, 2 picomoles, 20 nM) of FCE in 100 μl reactions containing 1X FCE buffer for 1 hour at 37°C. Following capping reactions, mRNA capping was measured using targeted RNase H cleavage and LC-MS.



     
    This product is related to the following categories:
    RNA Modification Products,
    RNA Reagents Products,
    RNA Synthesis Products
    This product can be used in the following applications:
    RNA Modification,
    RNA Analysis,
    In vitro Transcription for RNA Synthesis
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