In Vitro Selection and Characterization of a Light‐up DNA Aptamer for Thiazole Orange

A new DNA aptamer that binds to the target Thiazole Orange‐biotin (TO1‐biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin‐coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off‐rate binding. Using next‐generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three‐tiered G‐quadruplex motif for these families. This indicates that the TO1‐biotin DNA aptamer identified here uses a similar ligand‐binding topology to the original Mango RNA aptamer. This study explores the differences between RNA and DNA aptamers for the same target molecule. In vitro selection was used to identify a DNA version of the aptamer for Thiazole Orange. The resulting sequences were analyzed using computational tools to discover aptamer candidates, and several were characterized with biophysical techniques.