Fluorescence-based DNA binding assay

Fluorescence polarization assays were performed using a Synergy 4 microplate reader (BioTek) to measure DNA binding affinity. The 6-carboxy-fluorescein (FAM)-labeled double strand DNA probes (5 nM) were incubated with increasing amount of proteins (monomer concentration 0.8 nM to 13.25 μM) for 15 min in 20 mM Tris (pH 7.5), 5% (w/v) glycerol, 450 mM NaCl, 0.5 mM TCEP and 0.1 mg/ml BSA. GraphPad Prism software (version 7.0) was used to do curve fitting. K_D values were calculated as [mP] = [maximum mP] × [C]/ (K_D + [C]) + [baseline mP], where mP is millipolarization and [C] is protein concentration, and ΔmP = ([mP]—[baseline mP]). The reported mean ± SEM of the interpolated K_D values were calculated from two independent experiments performed in duplicate. We note that the mP amplitudes for the oligos containing 5hmC did not reach the same level as those of C- and 5mC-containing oligos (Supplementary Figure S3), and the lower limit of the binding affinity was estimated.