Binding Kinetics of CCHFV N Protein

Biolayer interferometry was used to monitor the binding affinities of wild type CCHFV N protein and its stalk domain with the 5’ untranslated regions (5’ UTR) of CCHFV S-segment derived mRNA, using the BLItz system (ForteBio Inc.), as previously reported [22 (link)–24 (link)]. Briefly, the biotin- 5’ UTR was synthesized and loaded onto high precision streptavidin biosensors (catalog # 18–5019, Forte Bio Inc.), as previously reported [20 (link)]. All reactions were carried out at room temperature in RNA binding buffer (20 mM Tris-HCl, pH 7.4, 80 mM NaCl, 20mM KCl, and 1mM DTT). After mounting the RNA, the biosensors were equilibrated in RNA binding buffer and then dipped in the purified protein solutions of either wild type N protein or stalk domain for the measurement of association kinetics. The reaction cycles were as follows: initial base line for 30 seconds, loading of biotinylated RNA on streptavdin biosensors for 120 seconds, base line for 30 seconds, association of protein with the RNA for 300s, followed by dissociation phase of 500 seconds. The kinetic parameters K_ass (association rate constant), K_dis (dissociation rate constant) and the binding affinities (Kd = K_dis/K_ass) were calculated with the help of inbuilt data analysis software (BLItZ Pro), as previously reported [22 (link)–24 (link)].