Structure-Guided Discovery of High-Affinity Methotrexate Aptamers for Electrochemical Drug Monitoring
- 1La Trobe University, Queensland University of Technology
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.
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BibTeX
@misc{sequeira2026structureguided,
title = {{Structure-Guided Discovery of High-Affinity Methotrexate Aptamers for Electrochemical Drug Monitoring}},
author = {M. B. Sequeira and T. C. Pimenta and R. Vieira and A. S. Beliaev and K. Alexandrov and S. F. Sousa and S. M. Silva and E. Eremeeva},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.06.757117},
url = {https://www.biorxiv.org/content/10.64898/2026.10.06.757117v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Structure-Guided Discovery of High-Affinity Methotrexate Aptamers for Electrochemical Drug Monitoring
AU - M. B. Sequeira
AU - T. C. Pimenta
AU - R. Vieira
AU - A. S. Beliaev
AU - K. Alexandrov
AU - S. F. Sousa
AU - S. M. Silva
AU - E. Eremeeva
PY - 2026
DA - 2026/10/06
PB - bioRxiv
DO - 10.64898/2026.10.06.757117
UR - https://www.biorxiv.org/content/10.64898/2026.10.06.757117v1
AB - 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.
ER -