Faustovirus Capping Enzyme |NEB酶试剂 New England Biolabs

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产品信息

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

Faustovirus Capping Enzyme |

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

Faustovirus Capping Enzyme |

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)

Faustovirus Capping Enzyme | 

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.

 
产品类别:
RNA Capping

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • M2081S     -20    
        Faustovirus Capping Enzyme M2081SVIAL -20 1 x 0.02 ml 25,000 units/ml
        FCE Capping Buffer B2181AVIAL -20 1 x 0.5 ml 10 X
        S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
        GTP N2080AVIAL -20 1 x 0.05 ml 10 mM
    • M2081L     -20    
        Faustovirus Capping Enzyme M2081LVIAL -20 1 x 0.1 ml 25,000 units/ml
        FCE Capping Buffer B2181AVIAL -20 1 x 0.5 ml 10 X
        S-adenosylmethionine (SAM) B9003SVIAL -20 1 x 0.1 ml 32 mM
        GTP N2080AAVIAL -20 1 x 0.25 ml 10 mM

  • 特性和用法

    单位定义

    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.

    反应条件

    1X FCE Capping Buffer
    Supplement with 0.5 mM GTP 和 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)

    贮存溶液

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

    热失活

    70°C for 10 minutes

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

  • 优势和特性

    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

  • 相关产品

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  • 参考文献

    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.

操作说明、说明书 & 用法

  • 操作说明

    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)
    3. Cap-0 synthesis using Faustovirus Capping Enzyme (FCE) (NEB #M2081)

  • 应用实例

    • Scaling of High-Yield In vitro Transcription Reactions for Linear Increase of RNA Production

FAQs & 问题解决指南

  • 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?