Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Ochratoxin A (OTA) is a mycotoxin with significant health implications.
• Aptamers offer specific molecular recognition for OTA detection.
• Understanding OTA-aptamer interactions is crucial for developing sensitive detection methods.

Purpose of the Study:

• To develop a rapid fluorescence anisotropy (FA) approach for analyzing ochratoxin A (OTA) and aptamer interactions.
• To investigate the influence of buffer compositions, particularly metal ions, on OTA-aptamer binding affinity.
• To identify key regions of the aptamer involved in OTA binding.

Main Methods:

• Utilizing the intrinsic fluorescence of OTA.
• Employing fluorescence anisotropy (FA) to monitor changes in OTA's rotational freedom upon aptamer binding.
• Performing aptamer mutation studies to map crucial binding regions.

Main Results:

• Specific binding of OTA to a 36-mer aptamer induced a significant increase in FA (approx. 0.160).
• The interaction was greatly enhanced by the simultaneous presence of Ca(2+) and Na(+).
• Binding affinity decreased over 18-fold with Ca(2+) alone and was lost without Ca(2+).

Conclusions:

• The FA approach provides a simple, rapid, and robust method for studying OTA-aptamer interactions.
• This method aids in optimizing buffer conditions and aptamer design for OTA detection assays.
• FA analysis is valuable for understanding aptamer-toxin binding dynamics and guiding aptamer engineering.