New England Biolabs, M0463S, yDcpS

yDcpS is a decapping enzyme that hydrolyzes m Related Categories RNA Modification Specification Unit Definition One unit is defined as the amount of yDcpS required to convert 50% of a 500 nM solution of the following 25-mer m7G-capped RNA to a 5’-diphosphorylated form in a total reaction volume of 20 μl in 1 hour at 37˚C: 5’-[m7Gppp]rGrUrArGrArArCrUrUrCrGrUrCrGrArGrUrArCrGrCrUrCrArA[3-6FAM]-3’ Reaction Conditions 1X yDcpS Reaction Buffer Incubate at 37°C 1X yDcpS Reaction Buffer 10 mM Bis-Tris-HCl 1 mM EDTA (pH 6.5 @ 25°C) Storage Buffer 300 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 70°C Specific Activity 20,000 units/mg Unit Assay Conditions 1X yDcpS Reaction Buffer with 500 nM m 7 G-capped 25-mer 3´-FAM RNA. After incubation at 37°C for 1 hour, yDcpS is inactivated at 70˚C for 15 minutes and decapping is measured by capillary electrophoresis. FAQ Q: Is yDcpS an alternative for Tobacco Acid Pyrophosphotase (TAP)? A: No. yDcpS leaves behind a 5′ diphosphate end upon decapping, which is not directly ligatable. TAP leaves behind a 5′ monophosphate that can be used for direct ligation using T4 RNA Ligase. Q: In the literature DcpS is reported to have no activity on transcripts longer that 25 nt. Is yDcpS from NEB active on longer transcripts? A: Yes. The decapping activity of yDcpS has been verified on in vitro transcripts up to 1.4 kb, as well as on total human RNA. Q: Is yDcpS active in Capping Buffer supplied with the Vaccinia Capping System (NEB #M2080)? A: No. The activity of yDcpS is sensitive to KCl and pH ≥7.5, both of which are found in Capping Buffer. Q: What is the target specificity of yDcpS? A: yDcpS has decapping activity on most guanosine-based caps, but no activity on A, C, or U caps. Cap0 and Cap1 structures are both decapped by yDcpS. Importantly, yDcpS does not remove desthiobiotin GTP or , m2,2,7G caps. Q: Is yDcpS 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: What are the preferred buffer conditions for yDcpS? A: The pH optimum for yDcpS is between pH 6-7. Phosphate buffer interferes with yDcpS activity. yDcpS activity is reduced in buffers containing ≥100 mM NaCl or ≥50 mM KCl. Q: Is yDcpS active at higher temperatures than 37˚C? A: Yes. yDcpS activity has been confirmed up to 50˚C.