Product Class: Other

FTO RNA Demethylase
cloned at NEB recombinant 37 No


Catalog #M0616

Product Introduction

  • FTO RNA Demethylase catalyzes the demethylation of N6-methyladenosine (m6A) on RNA to form adenosine.
  • FTO RNA Demethylase is an Fe(II)/alpha-ketoglutarate (αKG)-dependent dioxygenase that requires the presence of Fe(II), αKG, and sodium-L-ascorbate in the reaction mix for its demethylation activity.
  • 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.

Product Information

Description

FTO is named the fat mass and obesity-associated protein (FTO) due to its association with human obesity. This enzyme is a homolog of the Fe(II)/ alpha-ketoglutarate (αKG)–dependent AlkB dioxygenases and is an N6-methyladenosine (m6A) demethylase of eukaryotic mRNA. In vivo, FTO demethylates internal m6A and cap N6,2′-O-dimethyladenosine (m6Am) in mRNA and snRNA. FTO also mediates N1-methyladenosine (m1A) demethylation in tRNA. In vitro, FTO demethylates m6A, m6Am and m3U in ssRNA, as well as N6-methyl-deoxyadenosine (6mA) and 3-methylthymine (3mT) on ssDNA.
This product is related to the following categories:
RNA Library Prep for Illumina,
RNA Modification,

Properties & Usage

Unit Definition

A 25 µl reaction in FTO Reaction Buffer containing 100 ng of a 1700-mer N6A methylated Cluc mRNA and 0.5 ug of FTO RNA Demethylase incubated for 1h at 37 °C results in >50% demethylation of the substrate mRNA as determined by LCMS.

Reaction Conditions

50 mM Tris HCl, pH 7.5
1 mM αKG
1 mM sodium L-ascorbate
75 mM FE(II)
Incubate at 37 °C for 1 hour.

Storage Buffer

20 mM Tris-HCl
50 mM NaCl
50% Glycerol
0.1 mM TCEP

Heat Inactivation

No

Molecular Weight

Apparent: 59104.73 g/mol

Unit Assay Conditions

1 μg FTO RNA Demethylase is incubated with 100 ng of an Cluc mRNA substrate (1766 mer; 100% m6A), 50 mM Tris HCl, pH 7.5, 1 mM αKG, 1 mM sodium ascorbate, and 75 μM Fe(II) in 25 μL reaction at 37 °C for 1 hour. The reaction is then treated with 0.8 units of Proteinase K, Molecular Biology Grade (P8107) at 37°C for 1 h. The product RNA is purified using Monarch® RNA Cleanup Kit (T2040) then digested to nucleosides using the Nucleoside Digestion Mix (M0649). The extent of m6A demethylation is determined by monitoring the various adenosine species on an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135 XT MS Detector, on a Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 μm) with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5). The identity of each peak is confirmed by mass spectrometry. The relative abundance of each nucleoside is determined by the integration of each peak at 260 nm or their respective UV absorption maxima. 

Features

An RNA-seq product for N6-methyladenosine (m6A) mapping of RNA.

References

  1. Jia, G. et al. (2011). N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 7, 885 - 887.
  2. Fu, Y. et al. (2013). FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat Commun. 4, 1798.
  3. Gerken, T. et al. (2007). The obesity-associated FTO gene encodes a 2-oxoglutaratedependent nucleic acid demethylase. Science. 318, 1469-1470.
  4. Jia, G. (2007). Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO. FEBS Letters. 582 (23-24), 3313-3319.
  5. Li, Yixing. et al. (2019). The dynamics of FTO binding and demethylation from the m6A motifs. RNA biology. 1, 11.
  6. Zhang, X et al. (2019). Structural Insights into FTO’s Catalytic Mechanism for the Demethylation of Multiple RNA Substrates. Proceedings of the National Academy of Sciences 2019. 116 (8), 2919-2924.

Protocols, Manuals & Usage

Protocols

  1. Protocol for demethylation of an m6A-containing RNA by FTO RNA Demethylase (NEB #M0616)

FAQs & Troubleshooting

FAQs

  1. Are there any unreacted hm6A and f6A products at the end of the FTO RNA Demethylase reaction?
  2. What is the activity of FTO RNA Demethylase in other NEB buffers?
  3. What are Enzymes for Innovation?

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 [email protected].

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.

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