Structure-Guided Evolution of Broad-Spectrum Aptamers for Rapid Detection of Etomidate and Its Analogs

Abstract Etomidate (ETO) and its structural analogs (e.g., metomidate (METO) and propoxate (PROP)) are increasingly abused in e-cigarette juices (EJs), posing serious public health risks. The continuous emergence of new analogs to evade regulation has created an urgent need for detection methods resistant to substituent changes. Here, we performed library-immobilized SELEX using PROP as the target and identified a PROP-binding DNA aptamer (Y1A2) with a KD value of 4.5 μM. To improve its affinity and class cross-activity, we first characterized the structure−activity relationship of Y1A2 through systematic mutational scanning, truncation analysis, and circular dichroism spectroscopy. These studies revealed a conserved antiparallel G-quadruplex (G4) core. Guided by this structural map, we designed a focused secondary library fixing the G4 scaffold while randomizing several peripheral motifs; serial-SELEX against three ETO family targets yielded broad-spectrum aptamers with an order-of-magnitude improvement in affinity. The optimized aptamer (Y2A1) was integrated into a label-free thioflavin T displacement fluorescence assay, achieving limits of detection of 3.7−14.0 ng/mL for ETO and its analogs in 1% EJ within 10 min. This work provides a rational framework for improving aptamer affinity toward small-molecule families via structure-guided evolution, and delivers a practical tool for rapid screening of ETO-class drugs in seized e-cigarette sample.