Methods of optimization for KCE-based aptamer selection

The success of KCE-based aptamer selection relies on three distinct steps of pre-selection optimization: (i) preventing protein adsorption to the inner walls of the capillary, (ii) maximizing the protein-DNA separation, and (iii) determining an appropriate aptamer-collection window. To perform the first step of optimization, we have developed a simple pressure-based approach which can qualitatively characterize protein adsorption on capillary walls in CE1 o Conceptually, a short plug containing the protein solution is injected into the capillary and carried towards the detection point by applying a low pressure. A dual on-column detector is mimicked by repeating the experiment using the same capillary but a shortened distance to the detection window. The temporal propagation pattern of the protein, at each detection distance, is recorded and the degree of adsorption then analyzed by comparing peak areas and symmetry. This process can be repeated using different buffer additives2, dynamic or permanent coatings until optimal conditions are established. The remaining two steps of pre-selection optimization can be solved by fluorescently labelling the target protein so that its compatibility with KCE separation is maintained3 o By labeling the protein with Chromeo P503, we demonstrate that target detection in CE is markedly improved without significantly affecting the proteins electrophoretic mobility or ability to bind DNA. Thus, Chromeo-labelling can facilitate the accurate identification of both the protein and protein-aptamer complex, which is necessary for maximizing protein-DNA separation and selecting the aptamer collection window.

Target-specific considerations must also be optimized prior to commencement of KCE-SELEX. Exonuclease targets, such as Exonuclease I (E.coli), recognize the DNA library as a substrate to be degraded. As a result, the enzyme must be inactivated while still maintaining its native three-dimensional structure during aptamer collection. In addition, non-specific, or unwanted binding at the active site must be avoided, as collection of non-aptamers would limit the progress of KCE-SELEX. For Exol, a divalent metal chelating agent was found to effectively suppress library degradation through experimental optimization. In addition, a small oligonucleotide-based competitive inhibitor was found to bind to the active site with high affinity. This effectively eliminates any unwanted binding of the DNA library at the substrate binding site. The Exol-inhibitor complex can then serve as a target for aptamer development towards an allosteric site of the protein.