Systematic Evolution of Ligands by Exponential Enrichment to Sensor: Translating an Aptamer Into a Functional Electrochemical Sensor

Aptamers that undergo structural rearrangement upon ligand binding have increasingly been translated into sensing platforms such as electrochemical aptamer‐based biosensors. However, successful conversion from SELEX‐derived binders to functional sensors frequently requires extensive post‐SELEX engineering, which is typically performed in a largely empirical manner using nuclease digestion, truncation strategies, or computational predictions. Critically, these approaches do not explicitly account for ligand‐induced conformational change, a property widely proposed to be central to signal generation in structure‐switching aptamer sensors. To address this limitation, we evaluated whether solution‐based biophysical techniques—specifically isothermal titration calorimetry and NMR spectroscopy—could provide rational guidance for post‐SELEX engineering by directly reporting on global structural responses to ligand binding. This effort was enabled by the development of a new quantitative framework that, for the first time, allows the magnitude of ligand‐induced conformational change in aptamers to be experimentally measured. Using these methods, we established quantitative descriptors of aptamer structural remodeling and correlated them with downstream sensor performance. In doing so, we found that, while binding and ligand‐induced folding are necessary, sequence length emerges as the strongest predictor of successful sensor translation, likely reflecting its central role in mediating signal transduction.