Alpha-Helical Fragaceatoxin C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic Acid Analysis

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Nanopores are crucial for single-molecule DNA analysis and sequencing.
• Actinoporin proteins, like Fragaceatoxin C (FraC), are α-helical pore-forming toxins.
• Standard planar lipid bilayers are commonly used for nanopore reconstitution.

Purpose of the Study:

• To investigate the potential of Fragaceatoxin C (FraC) as a nanopore for DNA analysis.
• To engineer FraC for enhanced DNA detection capabilities.
• To explore the translocation of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) through FraC nanopores.

Main Methods:

• Reconstitution of engineered FraC into sphingomyelin-free standard planar lipid bilayers.
• Electrophysiological recordings to monitor DNA translocation events.
• Analysis of DNA-protein interactions based on nanopore current blockade signals.

Main Results:

• FraC was successfully reconstituted and demonstrated a funnel-shaped geometry suitable for DNA interaction.
• Engineered FraC could resolve homopolymeric stretches of single-stranded DNA (ssDNA) bases (C, T, A).
• Double-stranded DNA (dsDNA) translocated through the 1.2 nm constriction of FraC at high potentials, suggesting pore deformation.

Conclusions:

• Engineered FraC nanopores show promise for distinguishing ssDNA sequences.
• The ability of dsDNA to translocate through FraC opens possibilities for dsDNA analysis.
• FraC nanopores represent a novel tool for potential applications in DNA sequencing and analysis.