Equilibrium fluorescent RNA binding assays

Binding assays were carried out in 384-well plate format. Briefly, fluorophore-labeled hairpin RNA was diluted into binding buffer (25 mM MOPS pH 7.4, 150 mM NaCl, 5 mM DTT, 2 mM MgCl, 0.01% Triton X-100) to a concentration of 2 nM. The RIG-I protein construct of interest was then diluted into binding buffer over a 12-pt series of concentrations and mixed 1:1 with RNA samples (final RNA concentration of 2 nM) to a volume of 20 μl. Final RIG-I concentrations varied from 1.5 to 1500 nM. Samples were equilibrated at room temperature for 2 h. Fluorescence polarization was measured using a Biotek Synergy H1 plate reader. Samples were excited through a bandpass filter at 485/20 nM and fluorescence emission was measured through a bandpass filter at 528/20 nM. Polarization was calculated using the following Equation (2):

Where I_∥ is the intensity of the fluorescent light parallel to the plane of excitation, I_⊥ is the intensity of fluorescent light perpendicular to the plane of excitation and G is an empirically determined correction factor accounting for instrumental bias toward the detection of horizontally polarized light; in this case G = 0.87.

An individual experiment consisted of two replicates of each protein concentration for which polarization measurements were taken 3 times, yielding 6 values for each condition. The mean polarization values were then plotted against protein concentration and fit to a one-site total binding Equation (3):

Where y₀ represents the polarization value when [enzyme] = 0 nM, y_max represents the polarization achieved at a saturating enzyme concentration, and K_d is the dissociation constant. Three experiments were performed for each RIG-I construct, with each reported K_d value representing the mean and standard deviation across these experiments.