Measuring Aptamer Equilibria Using Gradient Micro Free Flow Electrophoresis

被引:40
作者
Turgeon, Ryan T. [1 ]
Fonslow, Bryan R. [1 ]
Jing, Meng [1 ]
Bowser, Michael T. [1 ]
机构
[1] Univ Minnesota, Dept Chem, Minneapolis, MN 55455 USA
基金
美国国家卫生研究院;
关键词
NONEQUILIBRIUM CAPILLARY-ELECTROPHORESIS; ZONE-ELECTROPHORESIS; BINDING CONSTANTS; REVERSE-TRANSCRIPTASE; CONTINUOUS SEPARATION; MOLECULAR-WEIGHT; AFFINITY; DEVICE; LIGANDS; ISOTACHOPHORESIS;
D O I
10.1021/ac902877v
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Gradient micro free flow electrophoresis (mu FFE) 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 mu FFE device, which separated ligand aptamer complexes from the unbound aptamer. The continuous nature of mu FFE 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.
引用
收藏
页码:3636 / 3641
页数:6
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