Advancing Kinetic Capillary Electrophoresis for High-Efficiency Screening of Oligonucleotide Libraries in Drug Discovery

Identifying protein binders is the first step in drug discovery. The combinatorial approach, in which a library of compounds is subjected to affinity screening against a target protein, is a major way for identifying protein binders. Oligonucleotide libraries constitute the largest source of material for such affinity screening. Selecting protein binders from such libraries requires a highly efficient method for separation of protein−oligonucleotide complexes from the excess of unbound oligonucleotides. Kinetic Capillary Electrophoresis (KCE) is a rapidly advancing technique in affinity applications. It reportedly has superior partitioning efficiency, but screening oligonucleotide libraries by KCE has many challenges which must be addressed before KCE can compete with conventional surface-based screening. The goal of my research is to transform KCE into a versatile technology for screening protein binders from oligonucleotide libraries. To overcome the nonbinder background issue in KCE-based partitioning, I introduce Ideal-Filter Capillary Electrophoresis (IFCE), where binders and nonbinders travel in opposite directions. While successfully implementing IFCE conditions to be compatible with physiological buffers, a remarkable partitioning efficiency of 109 is achieved, the highest recorded so far. Further, I develop the first quantitative characterization of all KCE-based partitioning modes for a diverse range of protein target sizes. This systematic analysis provides guidance for CE users on selecting appropriate KCE-based partitioning conditions for a given protein target. Next, I conduct the first experimental investigation into the influence of target concentration on binder selection, aiding researchers in identifying an appropriate range of target concentrations to prevent selection failures. Finally, I gather insights from all these works to demonstrate the first highly efficient KCE-based selection of protein binders from DNA-encoded library of small molecules (DEL). This pioneering achievement showcases the capabilities of KCE-based partitioning within the context of DEL-based drug discovery, marking a significant advancement in the field.