Single Molecule Dissociation of a Mycotoxin/Aptamer Complex in an α-hemolysin Nanopore

Abstract Mycotoxins analysis is essential for food safety since mycotoxins can cause severe toxic effects on humans and animals health. The aptasensor has been shown as a powerful detection platform for mycotoxins analysis. Aptamer, as a key recognition element in aptasensor for mycotoxin, has been speculated to fold from linear strand into elaborate tertiary structures, forming a selective binding pocket for mycotoxin. Direct observation of the binding or dissociation between mycotoxin and its aptamer at single-molecule level however remains a challenge. In this paper, ochratoxin A (OTA) aptamer with a poly (dC) 15 at 3' end is designed for the dissociation of OTA/aptamer complex and translocation in α-hemolysin (α-HL) nanopore. The large vestibule and the narrow constriction of α-HL allow the capture of OTA/aptamer complex and the dissociation, which can distinguish the conformation variation of OTA aptamer in the confined space. Under an appropriate condition, OTA/aptamer complex resulted in a distinguished type of current blockage (duration time > 200 ms) compared to the aptamer itself. The transient current oscillations were observed when OTA/aptamer complex was inside the vestibule of α-HL, followed by the escape to cis side (type I) or translocation to the trans side (type II). The single-molecule binding or dissociation properties between OTA and aptamer are affected by various factors like voltage, electrolytic buffer, as well as the existence of divalent cations. The result illustrated that biological nanopore may have the potential for uncovering the interaction mechanism between ochratoxin A and its aptamer.