title: Measuring aptamer equilibria using gradient micro free flow electrophoresis.
abstract: Gradient micro free flow electrophoresis (muFFE) was used to observe the equilibria of DNA aptamers with their targets (IgE or HIVRT) across a range of ligand concentrations. A continuous stream of aptamer was mixed online with an increasing concentration of target and introduced into the muFFE device, which separated ligand-aptamer complexes from the unbound aptamer. The continuous nature of muFFE allowed the equilibrium distribution of aptamer and complex to be measured at 300 discrete target concentrations within 5 min. This is a significant improvement in speed and precision over affinity capillary electrophoresis (ACE) assays. The dissociation constant of the aptamer-IgE complex was estimated to be 48 +/- 3 nM. The high coverage across the range of ligand concentrations allowed complex stoichiometries of the aptamer-HIVRT complexes to be observed. Nearly continuous observation of the equilibrium distribution from 0 to 500 nM HIVRT revealed the presence of complexes with 3:1 (aptamer/HIVRT), 2:1, and 1:1 stoichiometries.
authors.affiliation: Department of Chemistry, 207 Pleasant Street South East, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Turgeon
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Fonslow
authors.forename: Meng
authors.initials: M
authors.lastname: Jing
authors.forename: Michael T
authors.initials: MT
authors.lastname: Bowser
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D008832:Microchemistry
chemical_list: D052157:Aptamers, Nucleotide