FTO RNA Demethylase

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 LC-MS.

Reaction Conditions

50 mM Tris HCl, pH 7.5
1 mM αKG
1 mM sodium L-ascorbate
75 μM 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.