Surface Plasmon Resonance Binding Assay

A surface plasmon resonance (SPR) assay was performed to determine the binding affinities of aCPSF1 to poly-U-tract containing RNA on a BIAcore 8 K instrument (GE Healthcare) as described previously (Zhang et al., 2017 (link)) with minor modifications. A streptavidin-coated sensor chip SA (Series S Sensor chip SA, GE Healthcare) was first conditioned with three injections (10 µl min⁻¹) of buffer containing 1 M of NaCl and 50 mM of NaOH until a stable baseline was obtained. The 3′-biotinylated RNA was then diluted to 200 nM in binding buffer (20 mM HEPES-KOH, pH 7.5, 1 mM MgCl₂, 150 mM NaCl, 5% of glycerol, and 0.05% Tween 20) and immobilized in flow cell two at a flow rate of 10 µl min⁻¹ for 5 min. NaCl (500 mM) was then injected at 5 μl min⁻¹ to remove unbound RNA molecules until the response units (RU) reached a stable state. Mmp-aCPSF1 was twofold serially diluted from 1000–0 nM with binding buffer and continuously injected into flow cells with RNA immobilized and the control flow cell one without RNA of the sensor chip simultaneously at room temperature. The signal of flow cells with RNA was subtracted from that of flow cell one to eliminate nonspecific interactions. BSA was included as a negative control. The sensorgrams were analyzed using Biacore Insight Evaluation Software (version 1.0.5.11069, GE Healthcare).