Selection and Characterization of DNA Aptamers for Cytidine and Uridine

Abstract Cytidine and uridine are two essential pyrimidine ribonucleotides, and accurate detection of these nucleosides holds significant biological importance. While many aptamers were reported to bind purines, little success was achieved for pyrimidine binding. This study employs the library‐immobilization capture‐SELEX technique to isolate aptamers capable of selectively binding to cytidine and uridine. First, a selection was performed using a mixture of cytidine and uridine as the target. This selection led to the isolation of a highly selective aptamer for cytidine with a dissociation constant ( K d ) of 0.9 μM as determined by isothermal titration calorimetry (ITC). In addition, a dual‐recognition aptamer was also discovered, which exhibited selective binding to both cytidine and uridine. Subsequently, a separate selection was carried out using uridine as the sole target, and the resulting uridine aptamer displayed a K d of 4 μM based on a thioflavin T fluorescence assay and a K d of 102 μM based on ITC. These aptamers do not have a strict requirement of metal ions for binding, and they showed excellent selectivity since no binding was observed with their nucleobases or nucleotides. This study has resulted three aptamers for pyrimidines, which can be employed in biosensors and DNA switches.