4.6. Bio-Layer Interferometry Assay

The direct interaction between purified UP1 RNA-binding domain of the hnRNP A1 protein (N-terminal residues 1-196) and compounds was quantified with bio-layer interferometry (BLI) technique using OctetRED (ForteBio). hnRNP A1 was biotinylated in situ using an AviTag^TM sequence (GLNDIFEAQKIEWHE) (Avidity, LLC, Aurora, CO, USA) incorporated at the N-terminus of the hnRNP A1. Escherichia coli BL21 containing both biotin ligase and hnRNP A1 vectors were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and the protein was expressed for 4 h at 25 °C in the presence of 125 μM biotin. The bacteria were then lysed by sonication, and the resulting lysate was purified by immobilized metal ion affinity chromatography (IMAC) with nickel–nitrilotriacetic acid (Ni–NTA) resin followed by size exclusion chromatography (superdex s75, GE Healthcare). The purified hnRNP A1 at 0.1 mg/mL was immobilized on the streptavidin sensors (SA) overnight at 4 °C. The sensors were then blocked, washed and moved into wells containing various concentrations of the tested compounds in reaction buffer (20 mM Tris pH 8.5; 150mM NaCl; 0.5 mM TCEP; 5% DMSO and 10% glycerol).