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UBC Theses and Dissertations

The study of molecular interactions with capillary electrophoresis frontal analysis Qian, Cheng


Binding analysis has been a very important topic of many scientific research works for years. In Chapter 1, some most common binding analysis techniques were briefly reviewed. Six interesting variants of the CE-based methods are discussed including capillary electrophoresis frontal analysis (CE-FA). In Chapter 2, we demonstrated the development of a CE-FA method and its application in the affinity determination between a DNA binding domain and a drug candidate. The current data processing method of CE-FA mandates specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. Chapter 3 showed how significant errors occur when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real world applications becomes questionable. A mobility-based correction method was developed based on the flux of the ligand molecules. Conventionally, adding external pressure during the process of capillary electrophoresis separation has not been a common approach. While accelerating the separation, drawbacks including peak broadening and reduced resolving power are almost inevitable in pressure-assisted capillary electrophoresis (PACE). Frontal analysis (FA), with its unique characteristics, could potentially benefit from using external pressure while avoiding related drawbacks. In Chapter 4, the possible impact from pressure was studied mathematically and the physical process was simulated with COMSOL Multiphysics®. PACE-FA is applied to study the binding interactions between hydroxypropyl β-cyclodextrin (HP-β-CD) and small ligand molecules. In Chapter 5, we compared the binding constants obtained with electrospray ionization mass spectrometry (ESI-MS) and ACE. Two simulation programs were deployed to estimate the solvent impact on the binding interactions. In Chapter 6, a free solution method was developed for evaluating specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of non-specific binding without the need of reference compounds was proposed and tested. ESI-MS was first used to rapidly screen the small molecule candidates, then the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA).

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