Open tubular capillary electrochromatographic separations of peptides using G-quadruplex DNA stationary phases

This dissertation describes the investigation into the selectivity of G-quadruplex-forming DNA stationary phases towards amino acids and the separations of peptides using open-tubular capillary electrochromatography (OTCEC).; Series of dipeptides, including homodipeptides and alanyl dipeptides, fibrinogen peptides, and angiotensin peptides were separated using G-quadruplex DNA stationary phases in OTCEC. The stationary phases were constructed by covalently attaching the DNA oligonucleotide to the inner capillary surface. Three different G-quadruplex-forming oligonucleotides were investigated: the two-plane G-quadruplex-forming thrombin-binding aptamer, the four-plane analogue of the thrombin-binding aptamer, and a two-plane oligonucleotide identical to the thrombin-binding aptamer except for the replacement of guanine by thymine in the central bridging loop of the G-quadruplex structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin-binding aptamer but in a different sequence that does not allow G-quadruplex formation.; Separation results demonstrate that G-quadruplex DNA stationary phases exhibit selective interaction towards some amino acids and are able to separate closely related peptides that differ by one or two amino acids and peptides that differ by a single methyl group, under gentle and non-denaturing conditions.; Simulation modeling was also performed by collaborators Michael R. Goldsmith and Tatiana Prytkova in the Beratan research group at Duke University to study the structural conformations of the G-quadruplex oligonucleotide and to provide insights into the molecular interactions between the peptides and the oligonucleotide that give rise to the separation selectivity. The results demonstrate that the G-quadruplex oligonucleotide exhibits several possible interacting modes toward peptides, and that the interactions can be specific and often facilitated by the increased flexibility of the G-quadruplex structure at higher temperatures.