Some types of bacteria have the ability to punch holes into other cells and kill them. They do this by releasing pore-forming toxins (PFTs) that latch onto the cell’s membrane and form a pore. Punctured by multiple PFTs, the target cell self-destructs.
However, PFTs have garnered much interest beyond bacterial infections. The nano-sized pores that they form are used for “sensing” biomolecules, e.g., DNA or RNA, that pass through the nanopore like a string steered by a voltage, and its individual components (e.g., nucleic acids in DNA) give out distinct electrical signals that can be read out. In fact, nanopore sensing is already on the market as a major tool for DNA or RNA sequencing.
Molecular simulation of an engineered aerolysin pore (light blue color) embedded into a membrane bilayer (cream color) and translocating DNA (red color). [Chan Cao, EPFL]
Publishing (“Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores”) in Nature Communications, scientists led by Matteo Dal Peraro, PhD, an associate professor, at the École polytechnique fédérale de Lausanne (EPFL) have studied another major PFT that can be used effectively for more complex sensing, such as protein sequencing. The toxin is aerolysin, which is produced by the bacterium Aeromonas hydrophila, and is the founding member of a major family of PFTs found across many organisms.
One of the main advantages of aerolysin is that it forms very narrow pores that can tell apart molecules with much higher resolution than other toxins. Previous studies have shown that aerolysin can be used to “sense” several biomolecules, but there have been barely any studies on the relationship between aerolysin’s structure and its molecular sensing abilities, until now.
“Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure–function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance, and capabilities for sensing several biomolecules,” the investigators wrote.
“Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double β-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.”
The researchers first used a structural model of aerolysin to study its structure with computer simulations. As a protein, aerolysin is made up of amino acids, and the model helped the scientists understand how those amino acids affect the function of aerolysin in general.
Once they had a grasp of that relationship, the researchers began to strategically change different amino acids in the computer model. The model then predicted the possible impact of each change on the overall function of aerolysin.
At the end of the computational process, Chan Cao, PhD, a postdoctoral researcher and the leading author of this work, produced sixteen genetically engineered, mutant aerolysin pores, embedded them in lipid bilayers to simulate their position in a cell membrane, and carried out various measurements (single-channel recording and molecular translocation experiments) to understand how ionic conductance, ion selectivity, and translocation properties of the aerolysin pore are regulated on a molecular level.
And with this approach, the researchers finally found what drives the relationship between the structure and the function of aerolysin: its cap. The aerolysin pore isn’t just a tube that goes through the membrane, but it also has a cap-like structure that attracts and tethers the target molecule and pulls it through the pore’s channel. The study found that it is the electrostatics at this cap region that dictate this relationship.
“By understanding the details of how the structure of the aerolysin pore connects to its function, we can now engineer custom pores for various sensing applications,” said Dal Peraro. “These would open new, unexplored opportunities to sequence biomolecules as DNA, proteins, and their post-translational modifications with promising applications in gene sequencing and biomarkers detection for diagnostics.”
The scientists have already filed a patent for their sequencing and characterization of the genetically engineered aerolysin pores.
