Structural Insights into the Mechanism of High-Affinity Binding of Ochratoxin A by a DNA Aptamer.

A 36-mer guanine (G)-rich DNA aptamer (OBA36) is able to distinguish one atomic difference between ochratoxin analogues A (OTA) and B (OTB), showing prominent recognition specificity and affinity among hundreds of aptamers for small molecules. Why OBA36 has >100-fold higher binding affinity to OTA than OTB remains a long-standing question due to the lack of high-resolution structure. Here we report the solution NMR structure of the aptamer-OTA complex. It was found that OTA binding induces the aptamer to fold into a well-defined unique duplex-quadruplex structural scaffold stabilized by Mg²⁺ and Na⁺ ions. OTA does not directly interact with the G-quadruplex, but specifically binds at the junction between the double helix and G-quadruplex through π-π stacking, halogen bonding (X-bond), and hydrophobic interaction. OTB has the same binding site as OTA but lacks the X-bond. The strong X-bond formed between the chlorine atom of OTA and the aromatic ring of C5 is the key to discriminating the strong binding toward OTA. The present research contributes to a deeper insight of aptamer molecular recognition, reveals structural basis of the high-affinity binding of aptamers, and provides a foundation for further aptamer engineering and applications.