Fluorescence polarization assay for analysis of DNA binding

5′-FAM-labeled hemi-PT-DNA, labeled on one strand only, was synthesized and purified (Supplementary Table S1). Protein solutions were diluted serially using 2-fold dilutions (5 μM starting concentration, 16–20 dilutions) and mixed with a 5 nM final concentration of DNA probe in a Corning 3575 plate, using binding buffer of 20 mM Tris–HCl pH 8.0, 5% glycerol, 50 mM NaCl and 1 mM DTT. The mixture was incubated for 10 min at room temperature, and fluorescence polarization was measured at room temperature on a SpectraMax i3x (Molecular Devices) using 485/20 nm and 528/20 nm filters for emission and excitation, respectively. The dissociation constants (K_D) were calculated by fitting the experimental data (from two experimental replicates) to the following equation using GraphPad Prism software (version 6.0): [mP] = [maximum mP][C] /(K_D + [C]) + [baseline mP], and then the curve was replotted using percent saturation calculated as ([mP] – [baseline mP])/([maximum mP] – [baseline mP]), where mP is millipolarization and [C] is protein concentration. The binding experiments were performed under the same laboratory conditions.