Fluorescence polarization binding assays

The assays were performed at room temperature using a Neo2S plate reader (Biotek). The buffer for all the assays contained 20 mM Tris (pH 8.0), 150 mM NaCl, 10 mM DTT, 0.01% (v/v) NP-40, and 100 nM BSA. Oligonucleotides that included a polyadenylation site and the surrounding bases from the SV40 virus and a 6-carboxyfluorescein (6-FAM) label at the 5′ or 3′ end were used in direct binding assays. Oligonucleotides without a FAM label were used in competition binding assays. A 17-mer oligonucleotide with a 5′-end FAM label was used as the probe at a concentration of 1 nM in these experiments, and the protein was at 25 nM. For assays that involved titration with CPSF-30, various molar ratios of CPSF-30 (in complex with Fip1) were added to CPSF-160–WDR33. For other binding assays, 1 µM Fip1 was also included. All the mixtures were incubated on ice for 1 h and then transferred to 384-well plates at room temperature. Oligonucleotides were purchased from IDT. All titration experiments were carried out in triplicate.

Both the direct and competition binding experiments were fit to analytical equations (Wang 1995; Lundblad et al. 1996) using the optimize package from SciPy version 1.1.0 (Oliphant 2007). The fit to direct binding data took into account the depletion of the free probe during the titration. Statistical significance was tested by performing an F-test using both the global fit and individual fits of pairs of curves, with the stats package from SciPy version 1.1.0.