Encapsulating a single G-quadruplex aptamer in a protein nanocavity

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Biology

Background:

• The alpha-hemolysin (alphaHL) protein pore is a versatile tool with numerous biotechnological applications.
• Understanding single-molecule behavior is crucial for advancing molecular engineering and diagnostics.

Purpose of the Study:

• To develop a single-molecule manipulation system utilizing the alphaHL pore's nanocavity.
• To investigate the folding, unfolding, and translocation dynamics of thrombin-binding aptamer (TBA) G-quadruplexes within the nanocavity.

Main Methods:

• Utilized a single-molecule manipulation system based on the alphaHL protein pore.
• Employed noncovalent encapsulation of thrombin-binding aptamer (TBA) within the pore's nanocavity.
• Monitored changes in pore conductance to detect molecular events and used Tag-TBA for localization.

Main Results:

• Observed characteristic changes in pore conductance upon trapping the TBA G-quadruplex.
• Detected spontaneous unfolding of the G-quadruplex structure and translocation of unfolded DNA.
• Localized the G-quadruplex near the beta-barrel entry, observing molecular vibration and rotation.

Conclusions:

• The developed guest-nanocavity supramolecular system effectively probes single-molecule dynamics.
• This system provides insights into the folding and unfolding kinetics of G-quadruplex structures.
• Potential applications exist for understanding complex molecular processes at the single-molecule level.