1. Measuring Aptamer Equilibria Using Gradient Micro Free Flow Electrophoresis.
- Author
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Turgeon, Ryan T., Fonslow, Bryan R., Meng Jing, and Bowser, Michael T.
- Subjects
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ELECTROPHORESIS , *DNA , *LIGANDS (Chemistry) , *STOICHIOMETRY , *EQUILIBRIUM , *ELECTRIC fields , *SILICON - 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 aptainer-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/HlVRT), 2:1, and 1:1 stoichiometries. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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