Aptamers as a tool for protein analysis and discovery

Aptamers are synthetic affinity ligands in high demand for medicine and life sciences. Methods for aptamer discovery are tedious and time-consuming. The classical screening approach is low throughput. Usually, the most abundant sequences found in the final aptamer pool are experimentally screened for their binding affinity to the target which limits aptamer applications. I addressed the need for improved selection and screening strategies using Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECCEEM). First, I demonstrated that the use of NECEEM for partitioning of aptamers from non-aptamers, improves speed and simplifies the selection process. NECEEM-based partitioning introduced new options for (i) selecting aptamers with predefined binding parameters, (ii) selecting aptamers from non-amplifiable libraries of small molecules, and (iii) increasing the number of sequences in an aptamer pool. Second I used NECEEM for screening and characterization of the selected aptamers. NECEEM enables the measurement of equilibrium (Kd or Kb) and rate constants (k on and koff) from a single experiment, which can be accomplished in a matter of minutes. I modified the screening strategy by introducing a tandem approach of asymmetric PCR, a method for in-capillary mixing of reactants, and NECEEM. My novel high-throughput approach experimentally screens all potential binders, from an aptamer pool.;I demonstrated the potential of aptamers for protein discovery and analysis. The Aptamer-facilitated Biomarker Discovery (AptaBiD) procedure was developed to find and purify extracellular biomarkers. I developed Aptamer-facilitated Protein Isolation from Cells (AptaPIC) to select aptamers for a recombinantly expressed protein in a cell lysate. The selected aptamers are then used to purify the protein from the same lysate. Finally, I was able, for the first time, to measure and subtract an electric field associated effects on koff, and demonstrate that an electric field itself increases koff by several times when increased from 0-600 V/cm, for the studied protein-DNA pairs.