Product Class: Other

Faustovirus Capping Enzyme
NEBU 37 70 Heat SAM

GMP-grade coming soon.


Catalog #M2081

Product Introduction

  • 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
  • Getting ready to scale up RNA synthesis? Download our new technical note “Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production” for a generalized set of recommendations for synthesizing high yields of RNA.
 

Product Information

Description

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.



 
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Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit of Faustovirus Capping Enzyme is defined as the amount of enzyme required to convert 75 pmol of a 20-mer transcript to Cap-0 RNA in 30 minutes at 37°C.

Reaction Conditions

1X FCE Capping Buffer
Supplement with 0.5 mM GTP and 0.1 mM S-adenosylmethionine (SAM)
Incubate at 37°C

1X FCE Capping Buffer
50 mM Tris-HCl
5 mM KCl
1 mM MgCl2
1 mM DTT
0.02% Poloxamer 188
(pH 8 @ 25.0°C)

Storage Buffer

40 mM Tris-HCl
100 mM NaCl
50 mM Arginine
0.1 mM TCEP
50% Glycerol
pH 8 @ 25°C

Heat Inactivation

70°C for 10 minutes

Addition of EDTA to 5 mM is recommended to avoid RNA hydrolysis.

Features

  • 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

References

  1. Ramanathan, A. et al. (2016). Nucleic Acids Res . 44 (16), 7511–7526.
  2. Furuichi, Y., LaFiandra, A. & Shatkin, A. (1977). Nature. 266, 235-239.
  3. Schlee, M., Hartmann, G. (2016). Nat Rev Immunol . 16, 568-580.

Protocols, Manuals & Usage

Protocols

  1. Faustovirus Capping Enzyme FCE Standard Capping Protocol (NEB #M2081)
  2. One-pot synthesis of Cap-1 Synthesis with Faustovirus Capping Enzyme (FCE) and Cap 2´-O-methyltransferase (NEB #M2081, #M0366)

Application Notes

FAQs & Troubleshooting

FAQs

  1. What are Cap-0 and Cap-1?
  2. Can Faustovirus Capping Enzyme (NEB #M2081) use cap analogs in a capping reaction?
  3. Can Faustovirus Capping Enzyme (NEB #M2081) use GTP analogs in a capping reaction?
  4. Is Faustovirus Capping Enzyme sensitive to secondary structure?
  5. Is Faustovirus Capping Enzyme active at temperatures other than 37°C?

Quality, Safety & Legal

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Specifications

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Certificate Of Analysis

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Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

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Licenses

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