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Abstract
Non-covalent molecular interactions play crucial role in regulatory biological processes, such as gene expression, DNA replication, signal transduction, cell-to-cell interaction, and immune response. The molecular mechanisms of the action of many drugs are based on forming non-covalent molecular complexes with therapeutic targets. The formation of non-covalent molecular complexes is pivotal to many analytical techniques and devices used in research and disease diagnostics.
Capillary electrophoresis (CE) has been one of rapidly growing analytical techniques to study affinity interactions in recent years. Since CE features quick analysis, high efficiency, high resolving power, low sample consumption and wide range of possible analytes, it is beneficial for analysis of biomolecules and their interactions.
In my dissertation I propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods and their application to selection of binding ligands to specific targets and characterizing their binding parameters. KCE is defined as the CE separation of species, which interact under equilibrium or non-equilibrium conditions during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. In this proof-of-principle work, I present two KCE methods: Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and Sweeping Capillary Electrophoresis (SweepCE), mathematical models of the methods, and demonstration of their applications. The spectrum of their applications includes: (i) measuring equilibrium and rate constants from a single experiment, (ii) quantitative affinity analyses of proteins, (iii) measuring temperature in capillary electrophoresis, (iv) studying thermochemistry of affinity interactions, and (v) kinetic selection of ligands from combinatorial libraries.
I used NECEEM to select and characterize DNA aptamers for protein farnesyltransferase (PFTase). A single round of NECEEM-based selection was sufficient to obtain an aptamer with an equilibrium dissociation constant equals 0.5 nM. The entire selection procedure (excluding cloning and sequencing) took less than 5 hours and consumed only 10-16 moles (1 picogram) of the target protein. The NECEEM-based selection can be easily automated to facilitate mass production of aptamers, and used for discovery and characterization of drug candidates and the development of new diagnostic methods.
