High-Affinity Three-Way Junction Aptamers for Sensing Cationic Polymyxin Antibiotics

Polymyxins B and E (colistin) were historically the last-resort antibiotics for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their narrow therapeutic windows and significant nephrotoxicity necessitate rapid and precise detection methods. In this study, we employed a mixture of polymyxins B and E as targets to isolate DNA aptamers via the capture-SELEX method. Sequence analysis revealed two primary families, both characterized by three-way junction structures. The first family, represented by PM-2, exhibits comparable low-micromolar affinity for both polymyxins B and E and features a loopless three-way junction. In contrast, the PM-3 family demonstrates higher potency and selectivity, binding polymyxin B with a dissociation constant of 440 nM, approximately 10-fold stronger than its binding to polymyxin E, as determined by isothermal titration calorimetry. Binding assays under varying ionic strengths showed that affinity decreased with increasing salt concentration, suggesting that electrostatic interactions drive molecular recognition. Furthermore, thioflavin T fluorescence spectroscopy confirmed target-specific binding. Leveraging these distinct binding profiles, we developed a sensor array capable of discriminating between these two closely related antibiotics using a principal component analysis. These aptamers provide a robust foundation for the analytical detection and monitoring of polymyxin antibiotics in clinical settings.