Is the Volume Exclusion Model Practicable for Nanopore Protein Sequencing?

Area of Science:

• Biophysics
• Biotechnology
• Molecular Biology

Background:

• Nanopore technology offers potential for single-molecule protein sequencing.
• Current methods struggle to differentiate all 20 natural amino acids due to limited ionic current differences.
• Existing models do not fully explain observed current changes during nanopore sensing.

Purpose of the Study:

• To investigate the limitations of the traditional volume exclusion model in nanopore sensing.
• To propose a modified sensing model incorporating solution conductivity (σ') for improved peptide analysis.
• To enhance the development of nanopore-based protein sequencing.

Main Methods:

• Calculated and compared ionic current blockages for all 20 natural amino acids using aerolysin nanopores.
• Developed a modified solution conductivity (σ') parameter within the volume exclusion model.
• Conducted nanopore experiments using a short peptide (VQIVYK) in wild-type and mutant nanopores.

Main Results:

• Individual amino acid current blockages are insufficient for distinct identification.
• The traditional volume exclusion model inadequately explains current blockage contributions.
• Nanopore-peptide interactions significantly influence current changes, often dominating over simple volume exclusion.

Conclusions:

• A modified sensing model, including solution conductivity (σ'), is necessary for accurate nanopore peptide analysis.
• Factors beyond simple volume exclusion, such as enhanced nanopore-peptide interactions, are critical for interpreting current signals.
• This research provides crucial insights for advancing nanopore protein sequencing technologies.