New England Biolabs, M0607S, NudC Pyrophosphatase

This is an Enzyme for Innovation (EFI). EFI is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities. Related Categories RNA Modification Specification Unit Definition 1 µM of NudC hydrolyzes 200 µM or more NAD + into NMN + and AMP in 1X NEBuffer 3.1 and 5 mM DTT at 37°C for 30 min. Reaction Conditions 1X NEBuffer™ r3.1 Supplement with 5 mM DTT Incubate at 37°C NEBuffer™ r3.1 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 300 mM NaCl 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.9 @ 25°C Heat Inactivation 65°C for 10 minutes FAQ Q: Does NudC (NEB #M0607) decap other cap structures? A: NEB has found that NudC decaps model RNA containing a 5′ App- group (adenylated), G-capped mRNA with an adenosine as the initiating nucleotide at different efficiency relative to NAD+-capped RNA. It has been reported that that NudC hydrolyzes dinucleotides such as ADP-ribose, ADP-glucose and metabolic cofactors containing an ADP moiety, such as FAD (1) and Coenzyme A and acetyl Coenzyme A (unpublished results). Therefore, NudC can potentially decap RNA carrying a host of canonical and non-canonical initiating nucleotides not limited to NAD+/NADH. 1. Frick D.N. and Bessman M.J. (1995) J. Biol. Chem. 270,1529-1534. Q: Does NudC decap canonical G-capped RNA? A: NEB has found that NudC decaps G-capped RNA with an adenosine as the initiating nucleotide efficiently, with G-capped RNA with an initiating guanosine much less so. Q: Is it possible to use NudC to generate ligatible 5′ monophosphate RNA from non-canonical initiating nucleotides only? A: For applications where non-canonical cap structures are to be distinguished from G-capped RNA in a 5′ ligation-based workflow, one can pre-treat the RNA sample with yDcpS (NEB #M0463), followed by Quick CIP (NEB #M0525) and a RNA purification step to turn G-capped RNA and existing 5′ phosphorylated RNA to 5′hydroxyl RNA, which is not available for ligation. Q: Is NudC decapping activity affected by the structure of the RNA? A: Yes. It has been reported that NudC's rate of decapping is slower on RNA containing 5′ secondary structure (1). If the RNA is expected to contain substantial secondary structure, one can increase the enzyme concentration and/or increase the incubation time to achieve a higher level of decapping. 1. Höfer K. et al. (2016) Nat Chem Biol.12, 730-734. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.