Measuring Aptamer Equilibria Using Gradient Micro Free Flow Electrophoresis
- 7 April 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 82 (9), 3636-3641
- https://doi.org/10.1021/ac902877v
Abstract
Gradient micro free flow electrophoresis (μFFE) 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 μFFE device, which separated ligand−aptamer complexes from the unbound aptamer. The continuous nature of μFFE 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.Keywords
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