Study of molecular interactions by nonequilibrium capillary electrophoresis of equilibrium mixtures: Originations, developments, and applications.

Molecular interactions play a vital role in regulating various physiological and biochemical processes in vivo. Kinetic capillary electrophoresis (KCE) is an analytical platform that offers significant advantages in studying the thermodynamic and kinetic parameters of molecular interactions. It enables the simultaneous analysis of these parameters within an interaction pattern and facilitates the screening of binding ligands with predetermined kinetic parameters. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was the first proposed KCE method, and it has found widespread use in studying molecular interactions involving proteins/aptamers, proteins/small molecules, and peptides/small molecules. The successful applications of NECEEM have demonstrated its promising potential for further development and broader application. However, there has been a dearth of recent reviews on NECEEM. To address this gap, our study provides a comprehensive description of NECEEM, encompassing its origins, development, and applications from 2015 to 2022. The primary focus of the applications section is on aptamer selection and screening of small-molecule ligands. Furthermore, we discuss important considerations in NECEEM experimental design, such as buffer suitability, detector selection, and protein adsorption. By offering this thorough review, we aim to contribute to the understanding, advancement, and wider utilization of NECEEM as a valuable tool for studying molecular interactions and facilitating the identification of potential ligands and targets.