Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach

Abstract

By taking advantage of the intrinsic fluorescence of ochratoxin A (OTA), we present a fluorescence anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased fluorescence anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca 2+ and Na + , while the binding affinity of aptamer decreases more than 18-fold when only Ca 2+ exists, and the binding is completely lost when Ca 2+ is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamerbased assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design.

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer

“…Principle of detection of OTA using single fluorophore-labeled aptamer The applied 36-mer anti-OTA aptamers had good selectivity and displayed a K d about 50 nM in the binding buffer solution, as previously reported [21,23]. The binding affinity of the aptamer depended on the Ca 2+ or Mg 2+ , and proper ionic strength was also required for the high binding affinity [21,23].…”

“…The binding affinity of the aptamer depended on the Ca 2+ or Mg 2+ , and proper ionic strength was also required for the high binding affinity [21,23]. The previous study showed the optimized binding buffer solution contained 10 mM Tris-HCl (pH 8.5), 20 mM CaCl 2 , and 120 mM NaCl [21,23].…”

“…Fluorescence anisotropy (FA) analysis was performed on JASCO FP-8300 fluorescence spectrometer (Japan) by using the L-format configuration, following our previously reported procedure [23]. The experiments were performed at 20°C.…”

“…We investigated the effect of the fluorophore labeling on the binding affinity of the aptamer by using our previously established FA approach taking advantage of the intrinsic fluorescence of OTA [23]. OTA emitted fluorescence at 440 nm with an excitation at 375 nm in the binding buffer solution.…”

“…The recent emergence of the OTA-binding aptamers allows new approaches for OTA detection with great promise [21,22]. The DNA aptamer for OTA has a high binding affinity, and the dissociation constant reaches about 50 nM [21,23]. Many aptamer-based methods for OTA analysis recently have been reported [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38].…”