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

Abstract

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.

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A

“…In the absence of OTA, the FAM is distant from the BHQ1, resulting in high fluorescence intensity. In the presence of OTA, OTA specifically binds to the aptamer, inducing the aptamer folding into a duplex–quadruplex structure [ 26 ], and the fluorescence is quenched due to the close proximity of FAM and BHQ1. Therefore, OTA detection can be achieved by measuring the change in fluorescence intensity of the aptamer probe upon OTA binding.…”

“…In this work, taking advantage of aptamer and molecular beacon strategy, we developed a simple and rapid fluorescence method for OTA detection by using a high-affinity DNA aptamer against OTA, which has a duplex and G-quadruplex structure in the affinity complex [ 26 ]. The aptamer was modified with a fluorescein (FAM) and a black hole quencher (BHQ1) at the 5′ terminal and the 3′ terminal, respectively.…”

Highly sensitive and specific assessment of ochratoxin A in herbal medicines via activator regeneration by electron transfer ATRP

“…The aptamer binds more strongly to the target molecule (OTA) than to the cDNA. 34,35 Upon addition of the target molecule, the cDNA with the fluorescent label is shed.…”

Structural Basis of Pyridostatin and Its Derivatives Specifically Binding to G-Quadruplexes

“…Unlike the binding site of PyPDS/PDS, L 1 Pt­(dien) with a large aromatic plane and platinum unit binds to both G-quadruplex and duplex motifs of MYT1L with high spatial matching (Figure a,b), while the aromatic plane stacks at the G-tetrad via π–π stacking and the platinum unit interacts with the duplex region via groove binding. In contrast, the ochratoxin A (OTA) with a small organic plane and flexible scaffold binds to the duplex and other base regions of the OBA33 QDH (Figure c) . The spatial structure of the ligand itself is important for its binding site to DNA .…”

“…In contrast, the ochratoxin A (OTA) with a small organic plane and flexible scaffold binds to the duplex and other base regions of the OBA33 QDH (Figure 5c). 40 The spatial structure of the ligand itself is important for its binding site to DNA. 41 The spatial selective and adaptive matching between ligands and G4s is critical for the specific recognition of G4s by ligand.…”