Improving the Accuracy, Versatility, and Partitioning Efficiency of Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures
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Studying the affinity and the kinetics of biomolecular interactions is an essential task in biology and pharmacology. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a promising technique that allows the measurement of the equilibrium dissociation constant (Kd), and the rate constant of dissociation (koff), and can also be used as the partitioning method in selection of aptamers. There are three critical issues that have hindered the wide adoption of NECEEM by the analytical community: (1) poor resilience of the method against systematic errors, (2) limited versatility in terms of emulating physiological conditions, and (3) restricted partitioning efficiency in DNA aptamer selection. The goals of my research project were (i) to develop strategies for minimizing the systematic errors in NECEEM; (ii) to make NECEEM compatible with the use of physiological buffers; and (iii) to eliminate the restricted partitioning efficiency of NECEEM in aptamer selection. To improve the accuracy of NECEEM, I have developed an approach for simultaneous determination of both the Kd and the concentration of one of the interacting molecules, which eliminates errors caused by the presence of improperly folded molecules in samples. Further, I have developed an algorithmic approach for optimization of NECEEM, which takes the interrelation between its experimental parameters into account and minimizes the systematic error in an objective manner. To make NECEEM compatible with physiological buffers, I have uncovered the cause behind the poor detectability of DNA in phosphate buffered saline, and have created a pressure-assisted modification of NECEEM that overcomes this problem. Lastly, I have determined that the irregular electrophoretic migration of DNA in strong electric fields is caused by the ability of DNA to form unusually stable complexes with its counterions. Based on these findings, I have developed two independent approaches that increase the efficiency of NECEEM in selection of DNA aptamers.