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 reducedaffinity when used in other contexts. This can be especially problematicfor biomedical applications in which aptamers are exposed to samplematrices with distinctive chemical properties, such as blood, sweat,or urine. We present a high-throughput screening procedure for adaptingexisting aptamers for use in samples whose chemical composition differsconsiderably from the original selection conditions. Building on priorwork from our group, we have utilized a modified DNA sequencer capableof screening up to 10(7) unique aptamer mutants for targetbinding under the desired assay conditions. As an exemplar, we screenedall 11,628 single- and double-substitution mutants for a previouslyreported glucose aptamer that was originally selected in high-ionicstrength buffer and exhibited relatively low affinity in physiologicalconditions. After a single round of screening, we identified aptamermutants with similar to four-fold increased affinity in physiologicalconditions. Interestingly, we found that the impact of single-basesubstitutions was relatively modest but observed considerably greaterbinding improvements among the double mutants, highlighting the importanceof cooperative effects between mutations. This approach should begeneralizable to other aptamers and environmental conditions for arange of applications.