Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA

The detection of single molecules such as single-stranded DNA (ssDNA) and other small molecules through biological nanopores is a powerful approach for analyzing DNA sequences and DNA shapes, as well as for disease diagnostics. The fixed diameter of biological nanopores restricts the size of biomolecules translocated through them. Although some nano-pores such as ClyA, FraC, Phi29p, and gamma-hemolysin have been shown to translocate double-stranded DNA (dsDNA), identifying the difference between dsDNA and three-way junction DNA is difficult using these native biological nanopores. OmpG, a major outer membrane protein, forms a nanosized pore with 14 beta-strands. Here, we create a modified OmpG that expands and truncates beta-hairpins, allowing the generation of small or large nanopores compared to that of wild-type (WT) OmpG nanopores. To determine the pore diameters of modified OmpGs, the change in the current amplitude of the various modified OmpGs was measured in the presence or absence of poly(ethylene glycol) at different molecular weights. Finally, we demonstrated the detection of various structures of DNA (branched DNA) depending on the nanopore size using OmpG WT or mutated OmpG nanopores. Insights into the changes in pore diameters will be crucial to form precise pore diameters for the detection of various types of single biomolecules and for sequencing DNA, peptides, and proteins.

Automated summary

Detection of single molecules using biological nanopores is a powerful method for analyzing DNA sequences and shapes, as well as diagnosing diseases. However, distinguishing between double-stranded DNA and three-way junction DNA using native biological nanopores is challenging. This study created a modified OmpG nanopore to detect various DNA structures.