A High-Affinity but Low-Abundance Kanamycin Aptamer Reveals Hybridization-Limited Capture-SELEX

Kanamycin A, or simply referred to as kanamycin, is an aminoglycoside antibiotic with a narrow therapeutic window. Aptamers are useful recognition molecules for their detection and continuous monitoring. However, a short and high-affinity kanamycin aptamer that works under physiological conditions is still lacking. In this work, we revisited a previous aptamer selection done at pH 8, which had been abandoned due to poor sequence enrichment. Its top sequence, named KAN8-1, shows a <i>K</i>_d of 51 nM at pH 7.5 for kanamycin as measured by isothermal titration calorimetry, and its affinities to kanamycin A and B are similar. Using NMR spectroscopy methods, the KAN8-1 aptamer undergoes ligand-induced folding and likely has a better-defined structure compared to the KAN6-1 aptamer, which was highly enriched in the pH 6 selection. Using the KAN8-1 aptamer, a strand-displacement biosensor was developed, and it has a limit of detection of 0.9 μM with excellent selectivity. This sensor also has a similar performance in serum. The reason for the poor enrichment of KAN8-1 was attributed to its low hybridization efficiency and poor hybridization stability to the capture strand as demonstrated by a fluorescence titration assay and melting analysis, which indicated a limitation of the capture-SELEX method.