Directly investigating the interaction between aptamers and thrombin by atomic force microscopy.

Area of Science:

• Biochemistry and Molecular Biology
• Biophysics
• Nanotechnology

Background:

• Aptamers are nucleic acid molecules used for protein recognition, detection, and inhibition.
• Previous studies on thrombin-binding aptamers (15apt, 27apt) using atomic force microscopy (AFM) required chemical immobilization of thrombin.
• A need existed for methods to study aptamer-thrombin interactions without altering thrombin's native state.

Purpose of the Study:

• To develop and apply a new AFM method for directly investigating specific interactions between thrombin and its aptamers.
• To analyze the unbinding dynamics and dissociation energy landscapes of aptamer/thrombin complexes.
• To compare the interaction mechanisms of two different thrombin-binding aptamers (15apt and 27apt).

Main Methods:

• Utilized atomic force microscopy (AFM) to directly probe interactions between thrombin and aptamers.
• Developed a novel approach to avoid chemical immobilization of thrombin during AFM measurements.
• Analyzed aptamer/thrombin unbinding dynamics and dissociation energy landscapes.

Main Results:

• Successfully demonstrated a method for direct, label-free investigation of aptamer-thrombin interactions using AFM.
• Characterized the unbinding dynamics and dissociation energy landscapes, providing insights into interaction strength and mechanisms.
• Observed that despite structural differences in buffer, 15apt and 27apt exhibit similar underlying interaction mechanisms with thrombin.

Conclusions:

• The novel AFM method enables direct study of aptamer-protein interactions without chemical immobilization, preserving the native state of the protein.
• The study provides detailed insights into the biophysical mechanisms governing aptamer-thrombin binding.
• The findings suggest a conserved interaction principle between thrombin and its aptamers, irrespective of aptamer structure.