Three-dimensional selection of leptin aptamers using capillary electrophoresis and implications for clone validation.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has been used as a fast and efficient way to select aptamers against protein targets and offers the advantage of separating bound DNA from unbound DNA in a free solution three-dimensional environment. CE-SELEX was used to select aptamers against human leptin protein. Two methods used to validate the aptamers' binding affinity against the target were performed and gave differing results. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) yielded K(D) values in the high nanomolar range, whereas the fluorescence intensity method gave K(D) values in the low micromolar range. These results may suggest that aptamer validation must be carried out in a similar environment to that of the selection partitioning step and the environment in which the aptamer is intended to be used. We also note that affinity binding by fluorescence intensity using microplate readers may be limited to targets that have relatively low k(off) rates, systematic errors may occur when aptamers are validated using two different techniques, and the immobilization of smaller targets onto plate wells can affect the binding of the DNA, giving rise to lower binding affinities.