Biolayer Interferometry (BLI)

BLI was used to measure the binding affinities, association rates, and dissociation rates of compounds and r(CGG)₁₂. 5′-Biotinylated r(CGG)₁₂ (60 nM) was folded in 1 × Kinetics Buffer (ForteBio) by heating at 95 °C for 5 min and slowly cooling to RT. The RNA (200 μL aliquots) was then added to a black 96-well plate (Greiner Bio-One). The compound of interest at varying concentrations (200 μL aliquots) was dissolved in 1 × Kinetics Buffer. A sample with no compound was used as the background. All experiments were performed at 30 °C with agitation set to 1000 rpm.

Data analyses and curve fitting were completed using Octet Data Analysis, version 7.0. Experimental data were fitted using the 2:1 heterogeneous ligand (HL) curve fit. Global analysis of all data sets acquired for different compound concentrations, assuming reversible binding, was completed using nonlinear least-squares fitting.