Development of a thermal-stable structure-switching cocaine-binding aptamer

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Cocaine-binding aptamers are crucial for biosensing applications.
• Existing aptamer sequences have limitations in thermal stability.
• Ligand-induced structure-switching is a key mechanism for aptamer function.

Purpose of the Study:

• To develop a novel cocaine-binding aptamer variant with enhanced thermal stability.
• To maintain the structure-switching binding mechanism for biosensing utility.
• To improve the overall performance of cocaine detection systems.

Main Methods:

• Optimization of the DNA sequence in stem 2 of the aptamer based on nearest neighbor thermodynamic parameters.
• Isothermal titration calorimetry (ITC) to assess binding affinity.
• UV and fluorescence spectroscopy to confirm thermal stability (melting temperature, T_m).

Main Results:

• The new aptamer variant (OR3) exhibits significantly higher melting temperatures when bound to ligands.
• A T_m increase of 4°C for cocaine-bound aptamer and 9°C for quinine-bound aptamer was observed.
• The OR3 aptamer shows slightly tighter binding affinity compared to the existing sequence.

Conclusions:

• The OR3 aptamer variant demonstrates superior thermal stability while retaining essential structure-switching and binding functions.
• These enhanced characteristics make the OR3 aptamer a more robust and useful construct for cocaine sensing applications.
• The optimized sequence provides a foundation for developing next-generation aptamer-based biosensors.