New England Biolabs, M2081L, Faustovirus Capping Enzyme

Related Categories RNA Capping Specification Unit Definition One unit of Faustovirus Capping Enzyme is defined as the amount of enzyme required to convert 75 pmol of a 20-mer ppp-RNA to Cap-0 RNA in 30 minutes at 37°C. Reaction Conditions 1X Capping Buffer Supplement with 0.5 mM GTP and 0.1 mM S-adenosylmethionine (SAM) Incubate at 37°C 1X Capping Buffer 50 mM Tris-HCl 5 mM KCl 1 mM MgCl2 1 mM DTT (pH 8 @ 25°C) Heat Inactivation 70°C for 10 minutes Addition of EDTA to 5 mM is recommended to avoid RNA hydrolysis. FAQ Q: What are Cap-0 and Cap-1? A: Cap-0, commonly referred to as m7G cap or m7GpppN (where N signifies any ribonucleotide), is a N7-methyl guanosine connected by a 5′ to 5′ triphosphate linkage to the 5′ end of a transcript. Cap-dependent translation initiation, which is the primary mechanism of translation initiation in eukaryotes, requires cap 0 structure. 2′-O-methylation at the first position following the Cap-0 nucleotide generates Cap-1, which can also be represented by m7GpppNm-. Cap-1 has been shown to prevent inappropriate activation of antiviral innate immune receptors by endogenous transcripts in vivo. Q: Can Faustovirus Capping Enzyme (NEB #M2081) use cap analogs in a capping reaction? A: No. Faustovirus Capping Enzyme uses GTP and SAM to generate a cap structure on 5′ triphosphate RNA, typically a synthetic transcript generated by in vitro transcription. Cap analogs such as GpppG (NEB #S1407), m7GpppG (NEB #S1404), m7GpppA (NEB #S1405S) and anti-reverse cap analog (ARCA; NEB #S1411), are directly incorporated into the transcript by an RNA polymerase as the first nucleotide at the 5′ end. Q: Can Faustovirus Capping Enzyme (NEB #M2081) use GTP analogs in a capping reaction? A: 3′-modified GTP analogs such as 3′-Biotin-GTP (NEB #N0760S) and 3′-desthiobiotin-GTP (NEB #N0761S) are only weakly incorporated by Faustovirus Capping Enzyme. Vaccinia Capping Enzyme (NEB #M2080) is our recommendation for this use case. Q: Is Faustovirus Capping Enzyme sensitive to secondary structure? A: Yes. Secondary structures that limit 5′ end accessibility will reduce capping yield. Incubating capping reactions at higher temperatures or pre-heating the sample prior to adding capping enzyme can help improve capping of structured substrates. In severe cases, altering the sequence to the transcript to reduce secondary structure may be required. Q: Is Faustovirus Capping Enzyme active at temperatures other than 37°C? A: FCE maintains near-optimal capping activity up to 50°C. Capping activity is reduced at temperatures below 37°C, though more activity is seen with FCE than with VCE at the same molar concentrations.