Improving Aptamer Specificity with Novel Counterselection Strategies
Rosch, Jonah Cosmo
Aptamers are single-stranded oligonucleotide affinity reagents that are used for diagnostic and therapeutic applications. By adopting unique tertiary structures, aptamers can bind to a variety of targets, including inorganic materials, small molecules, proteins, and cell surface receptors, with nanomolar affinity. Aptamers are isolated by a process known as systematic evolution of ligands by exponential enrichment (SELEX), which relies on iterative exposure of a random library to a target to enrich the library with high affinity binders. To impart specificity during SELEX, the library is typically exposed to undesired individual secondary targets in successive rounds of selection. While aptamers with high affinity are relatively easy to obtain using various SELEX technologies, a high degree of specificity is much more difficult to engineer due to limitations on throughput and a lack of stringency imparted during iterative selection rounds between a target and each off-target. Failure to impose enough stringency in selection rounds can lead to the isolation of cross-reactive aptamers, which is especially undesirable when targeting biomolecules that are part of larger families that share sequence and structural homology. This dissertation summarizes our efforts to design a novel selection platform to consistently isolate high affinity and specificity aptamers first using the structurally similar platelet-derived growth factor (PDGF) family of proteins as a model system. Next, we applied this selection workflow to isolate RNA aptamers for two targets: the kinesin-12 coverstrand peptide, for applications as antimitotics, and serum albumin, to improve circulation time and bioavailability for therapeutic delivery of small interfering RNA (siRNA). Finally, we applied our selection strategies to whole cells, using custom CRISPR-engineered cell lines to isolate DNA aptamers for glucose transporter 1 (GLUT1).