There are many fluorescence-based applications that can be used to characterize molecular interactions. However, available methods often depend on site-specific labeling techniques or binding-induced changes in conformation or size of the probed target molecule. To overcome these limitations, we applied a ratiometric dual-emission approach that quantifies ligand-induced spectral shifts with sub-nanometer sensitivity. The use of environment-sensitive near-infrared dyes with the method we describe enables affinity measurements and thermodynamic characterization without the explicit need for site-specific labeling or ligand-induced conformational changes. We demonstrate that in-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules, including proteins, antibodies, and nucleic acids. Thereby, the described method is not limited to a subset of molecules since even the most challenging samples of research and drug discovery projects like membrane proteins and intrinsically disordered proteins can be analyzed.
Keywords: binding studies; biomolecular interaction; interaction analysis; spectral shift; thermodynamics.