Studying protein-DNA interactions using kinetic capillary electrophoresis

Protein-DNA interactions are essential to a cell as they control all vital cellular functions such as DNA replication, repair, recombination and transcription. In order to understand the dynamics of these fundamental biological processes, it is important to know kinetic parameters for all steps involved in the formation and dissociation of relevant DNA-protein complexes. We propose Kinetic Capillary Electrophoresis (KCE) as a conceptual platform for development of kinetic homogenous affinity methods for measuring kinetic parameters of such interactions. KCE is defined as the capillary electrophoresis (CE) separation of species that interact during electrophoresis. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. One of the advantages of KCE is that mathematical modeling is not necessary for some of them. Here, I i) present the theoretical bases of KCE and propose a multi-method KCE toolbox as an integrated kinetic technique, ii) develop a new KCE method with simplified mathematical approach, iii) introduce a predictive mixing parameter that is essential for diffusion-based mixing in general and to KCE in particular, iv) demonstrate the advantage of the proposed concept for studying complex protein-DNA interactions. Being based on CE separation, KCE is a powerful tool for testing different hypotheses of interactions and accurately calculating multiple binding parameters such as i) high-affinity (specific) and low-affinity (non-specific) interactions that recur within the same protein-DNA pair ii) disassembly of DNA-multiple proteins complexes. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and selecting affinity probes and drug candidates from complex mixtures.