Real time binding assays between peptides and purified DENV-2 S1 E protein were performed using biolayer interferometry on an Octet QK system (Fortebio, Menlo Park, CA). This system monitors interference of light reflected from the surface of a fiber optic sensor to measure the thickness of molecules bound to the sensor surface. Purified, recombinant, 80% truncated DENV-2 S1 E protein was obtained from Hawaii Biotechnology (Honolulu, HI). Peptides were N-terminally biotinylated with a 5:1 molar ratio of NHS-LC-LC-Biotin (Pierce/ThermoFisher, Rockford, IL) in PBS pH 6.5 at 4uC. Excess biotinylation reagent was removed using Pepclean C-18 spin columns (Pierce/ThermoFisher, Rockford, IL). Biotinylated peptides were coupled to kinetics grade streptavidin high binding biosensors (Fortebio, Menlo Park, CA) at several different concentrations. Sensors coated with peptides were allowed to bind to E protein in PBS with 0.02% (v/v) Tween-20 and 1 mg/ml BSA at several different E protein concentrations. Binding kinetics were calculated using the Octet QK software package, which fit the observed binding curves to a 1:1 binding model to calculate the association rate constants. E protein was allowed to dissociate by incubation of the sensors in PBS. Dissociation curves were fit to a 1:1 model to calculate the dissociation rate constants. Binding affinities were calculated as the kinetic dissociation rate constant divided by the kinetic association rate constant.