Fluorescence polarization assay for protein-RNA binding

The fluorescence polarization assay was performed as previously described (Chen et al., 2021 (link)). The reaction was prepared with 0–25 μM recombinant protein, 10 nM FAM-labeled [AG]₁₀ RNA, 1 mM AMP-PNP (Roche), 1 mM MgCl₂, 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol, and 1% DMSO (as a solvent of RocA) with or without 50 μM RocA/aglafoline. After incubation at room temperature for 30 min, the mixture was transferred to a black 384-well microplate (Corning), and the fluorescence polarization was measured by an Infinite F-200 PRO (Tecan). Under ADP + Pi conditions, 1 mM ADP (Fujifilm Wako Chemicals) and 1 mM Na₂HPO₄ were used as substitutes for AMP-PNP. The data were fitted to the Hill equation to calculate K_d values and visualized by Igor Pro v8.01 (WaveMetrics). The affinity fold change was calculated as the fold reduction in the K_d of RocA compared to the K_d of DMSO.