(320e) Improving Aptamer Specificity with Stringent Counterselection Methods

Aptamers are single-stranded oligonucleotide affinity reagents that are used for diagnostic and therapeutic applications. Due to their 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 prospective target to enrich for 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.

To address this problem, we have revised the SELEX workflow to include a competitive equilibrium step. Within the SELEX workflow, after an immobilized target is incubated with the nucleic acid library, it is exposed to all desired off-targets simultaneously, which is hypothesized to increase the stringency of the selection by providing direct competition for aptamer binding. The off-targets are present in free solution and flowed over the immobilized target using a peristaltic pump, allowing their concentration and exposure time to be easily tuned. When coupled with high throughput sequencing (HTS), this selection strategy can identify prospective aptamers that are enriched as a function of counterselection stringency. We are currently investigating the ability of this novel approach to isolate aptamers with improved specificity against structurally similar targets that are from the same family of proteins, using platelet-derived growth factors (PDGFs) as a model system. Our initial studies verify that, in the absence of stringent counterselection, we can isolate aptamers that bind PDGF-BB with nanomolar affinity but possess limited specificity for PDGF-BB over PDGF-AA, -CC, and -DD. Follow-up studies using the competitive equilibrium approach have aimed to increase the stringency of the counterselection to isolate nucleic acid sequences that are more prominently enriched compared to the original selection. Sequences obtained from the more stringent selection will undergo affinity and specificity tests to validate the effectiveness of the competitive equilibrium step. We anticipate isolating aptamers that bind to PDGF-BB with low nanomolar affinity and high specificity against the off-targets. Overall, we expect that these studies will provide the framework for isolating aptamers whose specificity can be tuned to any target by incorporating more effective counterselection steps.