Abstract
Methotrexate (MTX) is a narrow-therapeutic-index drug widely used in cancer and autoimmune disease treatment, for which delayed clearance can cause severe toxicity and requires close therapeutic drug monitoring. Here, we integrate high-stringency Capture-SELEX, biophysical characterization, molecular modeling, mutational validation, and electrochemical transduction to develop two structurally distinct MTX-binding DNA aptamer families. Full-length 3315 and 930 bound MTX with apparent KD values of 89 and 408 nM, respectively. Systematic truncation generated compact functional variants: 3315-55 retained 63 nM affinity and broad buffer tolerance, whereas 930-65 reached an apparent KD of 44 nM in 10 mM Mg2⁺, revealing markedly different ion dependencies. Biolayer interferometry confirmed rapid and reversible MTX recognition, while 3315 variants retained binding in serum-containing buffer. Computational analysis and site-directed mutagenesis identified distinct recognition architectures: a compact G-rich scaffold coupled to a central binding interface in 3315 and a localized Mg2+-stabilized binding pocket in 930. Methylene blue-labeled 3315-55 and 930-65 were integrated into electrochemical aptamer-based sensors, producing concentration-dependent responses to 100-1000 uM MTX in PBS, discriminating MTX from folic acid, and responding to MTX directly in undiluted human blood. Together, these results establish a structure-guided workflow linking aptamer discovery, mechanistic validation, and electrochemical translation, providing new molecular recognition elements and a foundation for future reagent-less MTX therapeutic drug monitoring.