title: A Fluorescence Polarization Biophysical Assay for the Naegleria DNA Hydroxylase Tet1.
abstract: The discovery of the 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (Tet) protein family was an important advancement in our understanding of DNA-modified epigenetics. Potent inhibitors of these proteins are greatly desired for both the understanding of the functions of these enzymes and to serve as eventual therapeutic leads. So far, the discovery of such small molecules with high affinity has been quite limited. Original tools to screen for activity are greatly needed in order to accelerate this process. Here we present a novel fluorescent probe, and the results of a fluorescence polarization-based binding assay forNaegleriaTet1, a homologue to mammalian Tet. A fluorescence polarization-based competition assay was also established and applied to the rapid and quantitative measurement of the binding affinity of the cofactor αKG and several known Tet1 inhibitors.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Marholz
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: RGENE Inc. , 953 Indiana Street, San Francisco, California 94107, United States.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado , Boulder, Colorado 80309, United States.
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
keywords: 5-hydroxymethylcytosine; Ten−eleven translocation (Tet) proteins; epigenetics; fluorescence polarization (FP)