Capture-SELEX-Derived Low-Nanomolar-Affinity Aptamers for Doxorubicin and Inhibition of Cellular Uptake

Anthracycline chemotherapeutics such as doxorubicin and daunorubicin remain indispensable in cancer treatment but possess narrow therapeutic windows, motivating the development of reliable molecular recognition elements for therapeutic drug monitoring, biosensing, and toxicity mitigation. Although an anthracycline aptamer has previously been obtained using immobilized daunorubicin, it lacks a well-defined secondary structure and exhibits poor affinity after truncation. Here, we employed capture-SELEX, which uses free target molecules in solution without immobilization, to isolate DNA aptamers that specifically recognize doxorubicin. The selection yielded three distinct sequence families with well-defined secondary structures and low-nanomolar affinities. Using intrinsic fluorescence quenching, isothermal titration calorimetry, and NMR spectroscopy, we rigorously distinguished specific aptamer recognition from nonspecific intercalation and demonstrated rapid, Mg²⁺-independent binding. The strongest aptamer, DOX-9, bound doxorubicin with a dissociation constant of 6 nM and also recognized daunorubicin with nanomolar affinity. Compared with the previously reported aptamer, the new sequences exhibit higher affinity, clearer structural features, and greater amenability to truncation. Importantly, they significantly inhibited doxorubicin uptake by HeLa cells, demonstrating potential as functional antidotes in addition to sensing elements. Together, these results establish a new generation of anthracycline aptamers and highlight capture-SELEX as an effective strategy for selecting aptamers against DNA-interacting small molecules.