Product Class: Other

mRNA Decapping Enzyme
NEBU 37 No


Catalog #M0608

Product Introduction

mRNA Decapping Enzyme decaps m7G-capped mRNA producing a 5′ monophosphate end.

  • Efficient replacement for Tobacco Acid Pyrophosphatase
  • Cap0 and Cap1 are removed with equal efficiency
  • Suitable for 5′ RLM-RACE and RNA-seq
  • 1 µl will decap 20 µg of a 1.7 kb mRNA in 15 minutes
 

Product Information

Description

mRNA Decapping Enzyme catalyzes the removal of 7-methylguanosine cap (m7G) from 5´ end of mRNA, producing 5′ monophosphate and releasing m7GDP(1). mRNA Decapping Enzyme is capable of decapping mRNAs of various lengths and removes both Cap0 and Cap1 structures with similar efficiency. mRNA Decapping Enzyme also converts 5′ triphosphate ends to 5′ monophosphate, albeit with reduced efficiency. 5′ monophosphorylated RNA can be exploited in a variety of downstream applications, including 5′ RNA Ligase-mediated RACE, RNA-seq, and 5′-> 3′ exonuclease digestion.

Comparison of mRNA Decapping Enzyme and Tobacco Acid Pyrophosphatase Decapping Activity.



(Panel A) A 500 nM solution of 25 nucleotide 5′ capped RNA was treated with either mRNA Decapping Enzyme (MDE) or competitor Tobacco Acid Pyrophosphatase (TAP). Both reactions were carried out in the same reaction volume and using each enzyme’s appropriate reaction buffer. While the concentration of a competitor’s TAP used is unknown, this comparison reveals 0.62 units MDE to have a similar level of activity relative to 1 µl of TAP. Decapping reaction products were analyzed by capillary electrophoresis. Note that in the negative control reaction there is a contaminating amount of triphosphorylated RNA of ~10% in the synthetic substrate used.
(Panel B) To test the impact of introducing length and termini heterogeneity into the reaction, 500 ng of total RNA for Jurkat cells was added while maintaining all other conditions unchanged. Decapping by MDE was unaffected, whereas TAP activity was markedly diminished, indicating that MDE is the more robust decapping enzyme under these test conditions.
 

 

Product Source

mRNA Decapping Enzyme from S.pombe is expressed as a His-tagged fusion in E. coli.
This product is related to the following categories:

Properties & Usage

Unit Definition

One unit is defined as the amount of mRNA Decapping Enzyme required to convert 50% of a 500 nM m7G-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

References

  1. Paquette, DR., et. al (2018). RNA. 24 (2), 251-257.
  2. Grudzien-Nogalska, E., and Kiledjian M (2017). Wiley Interdiscip Rev RNA. 8, e1379.

Protocols, Manuals & Usage

Protocols

  1. Standard Decapping Protocol (NEB #M0608)

FAQs & Troubleshooting

FAQs

  1. Does mRNA Decapping Enzyme remove 5′ triphosphates?
  2. Is mRNA Decapping Enzyme activity sensitive to RNA secondary structure?
  3. Does mRNA Decapping Enzyme have a preference for longer or shorter RNA?
  4. Is mRNA Decapping Enzyme active in other NEB reaction buffers?
  5. Does mRNA Decapping Enzyme require the presence of metals for activity?
  6. Is mRNA Decapping Enzyme active at higher temperatures?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

This product is for in vitro research use only.

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