Exploring New Nanopore Sensors from the Aerolysin Family

Abstract Aerolysin‐like proteins are a family of pore‐forming toxins with potential biotechnological applications as nanopore sensors for biomolecular detection and sequencing. Despite their conserved structural fold, the low sequence identity complicates sequence alignment, limiting the understanding of their pore structure and properties. Here, the pore structures of three family members – Clostridium perfringens epsilon toxin (ETX), Laetiporus sulphureus lectin (LSL), and Bacillus thuringiensis parasporin‐2 – are analyzed and compared to aerolysin and assess their single‐strand DNA (ssDNA) sensing capabilities through in silico methods. ETX is further characterized experimentally, revealing three distinct pore conformations, each with specific open pore currents, only one of which translocates ssDNA. Notably, ETX exhibited higher current blockage depth compared to aerolysin during ssDNA translocation, indicating a higher sensitivity for molecular sensing. The findings open new avenues for improving and diversifying nanopore capabilities in molecular sensing.