High-Throughput Strategy for Enhancing Aptamer Performance across Different Environmental Conditions

Aptamers selected under specific environmental conditions (e.g., pH, ion concentration, and temperature) often exhibit greatly reduced affinity when used in other contexts. This can be especially problematic for biomedical applications in which aptamers are exposed to sample matrices with distinctive chemical properties, such as blood, sweat, or urine. We present a high-throughput screening procedure for adapting existing aptamers for use in samples whose chemical composition differs considerably from the original selection conditions. Building on prior work from our group, we have utilized a modified DNA sequencer capable of screening up to 107 unique aptamer mutants for target binding under the desired assay conditions. As an exemplar, we screened all 11,628 single- and double-substitution mutants for a previously reported glucose aptamer that was originally selected in high-ionic strength buffer and exhibited relatively low affinity in physiological conditions. After a single round of screening, we identified aptamer mutants with ∼four-fold increased affinity in physiological conditions. Interestingly, we found that the impact of single-base substitutions was relatively modest but observed considerably greater binding improvements among the double mutants, highlighting the importance of cooperative effects between mutations. This approach should be generalizable to other aptamers and environmental conditions for a range of applications.