| Aptamers are oligonucleotides selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) method that bind to a target with high affinity and specificity based on the three dimensional conformation they adopt. Selected for targets ranging from small molecules to proteins or whole cells, aptamers may similarly assume diverse roles such as drug delivery vehicles, decoys, inhibitory therapeutics, and affinity separation probes. This work investigates aptamers as inhibitory drugs and affinity ligands in two bioanalytical applications.;In the first set of studies, an aptamer was selected to the peptide anticoagulant bivalirudin to serve as an antidote to the drug in instances of severe patient bleeding. The drug was immobilized on a monolithic column and binding sequences were eluted by salt gradient. The elution profile was compared to that of a blank column (no drug), and fractions with a chromatographic difference between drug-immobilized and blank counterparts were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified which demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer displayed a dose-dependent reduction of the clotting time in buffer, with a 20 muM aptamer concentration achieving a nearly complete antidote effect. A DNA microarray was used to truncate two of the aptamers to decrease the cost of aptamer synthesis for medical applications.;The second group of experiments tested a simple aptamer-based square capillary cell affinity chromatography system for selective capture of target cancer cells from a flowing suspension. Aptamers were immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which was controlled by adjusting the aptamer concentration. The device captured target leukemia cells in higher amounts than nontarget cells under a variety of conditions, and the capture efficiency was optimized to retain >90% of cells. In addition, the system was used to capture two colon cancer cell lines by their respective aptamers at high efficiency. The capillary system could also detect stained cancer cells spiked in blood by imaging the length of the tubing and counting the fluorescent captured cells. |
