Nanopore force spectroscopy of aptamer-ligand complexes

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Aptamers are crucial in molecular recognition, but their binding dynamics and stability require precise characterization.
• Nanopore-based dynamic force spectroscopy offers a unique platform for probing molecular interactions at the single-molecule level.

Purpose of the Study:

• To investigate the stability and binding characteristics of aptamer-ligand complexes using nanopore dynamic force spectroscopy.
• To determine dissociation constants and explore ligand specificity for aptamers targeting ATP and thrombin.

Main Methods:

• Utilized backward translocation technique with α-hemolysin nanopores to analyze ATP-binding aptamer interactions.
• Employed nanopore force spectroscopy, varying buffer potassium content, to study thrombin-binding aptamer interactions.
• Investigated aptamer conformational dynamics and ligand-dependent binding affinities.

Main Results:

• Determined the dissociation constant (Kd) of the ATP-binding aptamer to be approximately 0.1 mM.
• Observed distinct binding patterns for AMP, ADP, and ATP ligands, indicating ligand specificity.
• Identified conformational switching in the aptamer before ATP binding and detected thrombin presence.
• Highlighted limitations for aptamers with strong secondary structures and high-affinity complexes (nanomolar Kd).

Conclusions:

• Nanopore dynamic force spectroscopy is a valuable tool for characterizing aptamer-ligand complex stability and binding kinetics.
• The study provides insights into aptamer conformational dynamics and ligand recognition mechanisms.
• Methodological limitations exist for certain aptamer types and high-affinity interactions.