Quantifying Protein-Dna Interactions By Kinetics Exclusion Assay

Protein-DNA interactions are vital components of the regulatory cellular machinery in health and disease. Quantifying these interactions is essential for understanding their origin, specificity, and the role they play in regulating intricate cellular processes. Although numerous laboratory techniques have been developed to study the intricate interactions between proteins and DNA, each one of them has limited utility regarding applications. For better quantification of these important interactions, our work focused on using the Kinetics Exclusion Assay (Kinexa) technology. Our strategies included using either DNA or protein for constructing the titrant-coated solid phase. The captured constant binding partner was identified by using intercalating fluorescent probes binding DNA molecules or fluorescently-labeled probes targeting proteins. The proposed methodologies provide a unique way to obtain true solution-phase measurements since the molecules in solution are unmodified and the measurement process itself has a minimal impact on equilibrium.