De novo design of DNA aptamers that target okadaic acid (OA) by docking-then-assembling of single nucleotides

Okadaic acid (OA) is a diarrhetic shellfish poison widespread in ocean, so its detection is of great significance to seafood safety. Because of good sensitivity and low cost, biosensors using nucleic-acid aptamers as the recog-nition molecules are emerging as an important detection tool. However, the traditional SELEX screening method for acquiring OA high-affinity aptamers is time-and resource-intensive. Alternatively, here we developed a de novo design method based on the 3D structure of a target molecule, such as OA. Without experimental screening, this method designs OA aptamers by a computational approach of docking-then-assembling (DTA) of single nucleotides (A, C, G and T) as: (1) determining the high-affinity nucleotide binding sites of the target molecule via saturated molecular docking; (2) assembling the bound nucleotides into binding units to the target molecule; (3) constructing full-length aptamers by introducing stabilizing units to connect these binding units. In this way, five OA aptamers were designed, and microscale thermophoresis (MST) experiments verified that their Kd values are in the range of 100-600 nM; and one of them (named 9CGAT_4_a) could specifically bind to OA with low affinities for the other three marine biotoxins. Therefore, this study provides high-affinity and specific aptamers for the development of OA biosensors, and presents a promising de novo design method applicable to other target molecules.