Probing Streptavidin-Biotin Interaction and Complex Structure at the Single-Molecule Level Using a Self-Crowded...

Area of Science:

• Biophysics
• Analytical Chemistry
• Molecular Biology

Background:

• Probing biomolecular interactions is crucial for understanding biological systems and drug development.
• Current methods face challenges in label-free, single-molecule detection of small molecules under physiological conditions.
• Nanopipette-based nanopore sensing offers a cost-effective solution for label-free single-molecule analysis in solution.

Purpose of the Study:

• To improve nanopipette sensitivity for detecting small molecule interactions.
• To apply this enhanced method to study the stoichiometry and structure of molecular complexes.
• To investigate the influence of external factors on molecular complex formation.

Main Methods:

• Utilized a self-crowding technique to enhance nanopipette sensitivity.
• Employed streptavidin-biotin interaction as a model system for studying molecular binding.
• Analyzed unique multipeak ionic current spikes generated during molecular interactions.

Main Results:

• Observed unique multipeak ionic current spikes indicating complex formation.
• Demonstrated that peak characteristics correlate with biotin conjugate size, revealing stoichiometry and surface contour.
• Showed that crowding levels and concentration ratios influence complex stoichiometry.

Conclusions:

• The enhanced nanopipette approach provides high spatial resolution for characterizing label-free small molecule interactions.
• This method is promising for studying molecular interactions in crowded environments.
• The technique enables high-resolution structural characterization of molecular complexes.