New England Biolabs, M0608S, mRNA Decapping Enzyme

mRNA Decapping Enzyme decaps m Related Categories RNA Modification Specification Unit Definition One unit is defined as the amount of mRNA Decapping Enzyme required to convert 50% of a 500 nM m 7 G-capped substrate to a 5´-monophosphorylated form in a total reaction volume of 20 μl in 1 hour at 37˚C. Reaction Conditions 1X mRNA Decapping Enzyme Reaction Buffer Incubate at 37°C 1X mRNA Decapping Enzyme Reaction Buffer 50 mM Tris-HCl 50 mM Ammonium Chloride 5 mM MgCl2 1 mM DTT 0.1% Poloxamer 188 (pH 7.5 @ 25°C) Storage Buffer 300 mM NaCl 10 mM Tris-HCl 0.1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No FAQ Q: Does mRNA Decapping Enzyme remove 5′ triphosphates? A: Yes, albeit with lower efficiency. Under unit assay reaction conditions, 10-fold more enzyme was required to achieve 10% conversion of a triphosphorylated 25-nucleotide RNA substrate to the monophosphate form than was required to fully decap the same 25-nucleotide substrate with a cap. A 100-fold excess of enzyme only achieved 50% conversion. For conversion of triphosphate to monophosphate, RppH (NEB #M0356) is a superior option. Q: Is mRNA Decapping Enzyme activity sensitive to RNA secondary structure? A: Yes. Recessed or blunt 5′ ends are decapped less efficiently than single-stranded or 5′-overhanging ends. However, structural interference can be overcome by use of more enzyme, higher incubation temperatures or longer incubation times. Q: Does mRNA Decapping Enzyme have a preference for longer or shorter RNA? A: mRNA Decapping Enzyme has not shown obvious preferences for long or short RNA. When moles of RNA ends are equal, both a 25-nucleotide and a 1.7 kb RNA are decapped with the same efficiency. Q: Is mRNA Decapping Enzyme active in other NEB reaction buffers? A: mRNA Decapping Enzyme is ~50% active in rCutSmart® Buffer, NEBuffer r1.1, and ThermoPol™ Buffer. Q: Does mRNA Decapping Enzyme require the presence of metals for activity? A: Yes, magnesium is required for decapping activity. Q: Is mRNA Decapping Enzyme active at higher temperatures? A: Yes. Full decapping activity is observed up to 45°C, and >50% reduced activity is observed up to 55°C. Care should be taken to reduce incubation times at elevated temperatures to avoid metal-dependent RNA hydrolysis.