Fluorescence-Based Biosensor to Quantify Small Molecule Binding Kinetics with Target Spatial Resolution

Abstract

We present a novel method for the measurement of small molecule binding kinetics and demonstrate how this approach is used to detect analyte binding to target proteins with spatial resolution. Proteins of interest are immobilized onto DNA-functionalized gold microelectrodes by conjugation to single-stranded DNA (using generic conjugation methods) and then hybridization to complementary DNA coupled on the biosensor surface. The fluorophore, carried by the surface-coupled DNA, serves as the reporter dye for the assay. The binding of a small molecule analyte in the vicinity of the fluorophore is observed, whereby rate of fluorescence signal change reflects the corresponding association and dissociate rates of the analyte from its target protein. An extension to this standard assay to enable the site-specific binding readout involves the co-immobilization of a target-specific tool compound. In this non-classical competition assay, the fluorescence signal of the fluorophore is impacted by proximity to the tool compound rather than the protein, typically observed as a signal quenching when the tool compound is displaced from its target binding site. An analyte concentration-dependent quenching of the fluorescence signal is a positive readout for tool compound displacement, and thus confirmation of site-specific binding. Furthermore, the actuation of the loaded DNA nanolevers, by applying alternating electric fields to the microelectrodes, is impacted by the hydrodynamic drag incurred by the protein target, and correlates to the target protein's Stokes diameter. Therefore, in addition to titration and flow experiments that yield KD values and kinetics (ka and kd) parameters for analyte to target binding, real-time binding-induced conformational changes can be simultaneously measured. Here, we demonstrate the range of applicability of this technique by presenting examples for the analysis of conformational changes and allosteric regulation in proteins - such as kinases and STING.