Binding Affinity of psPIWI-RE

Fluorescence polarization assays were performed manually in black 96 microplate as previously described (Miyoshi et al. 2016 (link)). Each well (100 μL) contained 5 nM 3′-6-FAM-labelled oligonucleotides and varied concentrations (0–1.5 μM) of PsPIWI-RE in reaction buffer (20 mM Tris–HCl (pH 7.5), 250 mM NaCl, and 5 mM MgCl₂) for 30 min at 37 °C. The polarisation values were measured with excitation at 494 nm, emission at 522 nm in a SpectraMax M5e reader (Molecular Devices, CA, USA). The data were fitted to a nonlinear regression curve using the one-site-specific binding function in GraphPad Prism 8. All experiments were performed in triplicate.