title: Analysis of DNA in Phosphate Buffered Saline Using Kinetic Capillary Electrophoresis.
abstract: Kinetic capillary electrophoresis (KCE) methods are useful in the study of kinetics and equilibrium properties of interactions between DNA and its binding partners (ligands). KCE experiments are typically performed in a narrow set of "conventional" low-conductivity run buffers while DNA-ligand interactions in biological systems occur in physiological fluids, characterized by high ionic strengths. The nature and ionic strength of the buffer, in which DNA-ligand interaction occurs, can significantly influence the binding. Therefore, KCE experiments meant to study such interactions would greatly benefit if they could be performed in physiological buffers, such as phosphate buffered saline (PBS). No previous KCE studies of DNA used PBS as the run buffer. Here, we test the feasibility of using PBS as a KCE run buffer for analysis of DNA and show that its usage under standard KCE conditions renders DNA undetectable. We uncover the causes of this previously unreported detrimental effect and come up with a modification of KCE which allows one to overcome it. We apply the modified KCE method to an experimental model of a platelet-derived growth factor (PDGF) protein and its DNA aptamer, which was selected in PBS, and show that the results obtained in PBS run buffer are much closer to previously reported values than those which were obtained with a conventional low-conductivity capillary electrophoresis (CE) buffer.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors.forename: Mirzo
authors.initials: M
authors.lastname: Kanoatov
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D002021:Buffers; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary; D007700:Kinetics; D008024:Ligands; D010710:Phosphates / Q000737:chemistry; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D052157:Aptamers, Nucleotide; D001896:Boron Compounds; D002021:Buffers; D008024:Ligands; D010710:Phosphates; D010982:Platelet-Derived Growth Factor; D004247:DNA