Single molecule nanocontainers made porous using a bacterial toxin

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Vesicle encapsulation enables biofriendly immobilization for single-molecule studies.
• Previous methods using DMPC vesicles for porous bioreactors had limitations.
• Bacterial pore-forming toxins offer an alternative strategy for creating permeable vesicles.

Purpose of the Study:

• To develop a novel method for creating porous vesicles using alpha-hemolysin.
• To demonstrate the utility of these protein-pore vesicles for studying molecular interactions and reactions.
• To compare the stability and functionality of these vesicles with previous DMPC-based systems.

Main Methods:

• Utilized alpha-hemolysin, a bacterial pore-forming toxin, to create pores in vesicles.
• Encapsulated RNA molecules to study magnesium ion exchange and response to buffer changes.
• Co-encapsulated helicase protein and DNA to investigate translocation activity.
• Performed flow measurements to assess molecular exchange and reaction kinetics.

Main Results:

• Protein-based pores allowed magnesium ion exchange while retaining encapsulated RNA.
• Encapsulated RNA molecules showed rapid responses to external buffer condition changes.
• Demonstrated ATP-fueled DNA translocation by E. coli Rep helicase within vesicles, with enhanced cycle numbers due to small vesicle volume.
• The alpha-hemolysin pores exhibited stability over a wide range of temperatures, unlike DMPC vesicles.

Conclusions:

• Alpha-hemolysin-based porous vesicles provide a robust platform for bioassays and single-molecule studies.
• This method enables controlled molecular exchange and enhances the efficiency of enzymatic reactions within confined volumes.
• The stability and potential for engineering of these protein pores offer significant advantages for future applications in synthetic biology and nanotechnology.