Nanopore-Based Analysis of Conformational Heterogeneity of Nucleic Acids using a Gamma-Hemolysin Protein Channel

Abstract

Protein nanopores have been used to stochastically detect and characterize analytes for chemical reactions, biomolecule analysis, or biomedical applications. The detection sensitivity and specificity of the targeted analyte are significantly affected by both physical and chemical interactions between the analyte and the selected protein pore. We have demonstrated that a γ-hemolysin (γ-HL) protein channel (HlgB-HlgC) can assemble into an octameric pore on a planar lipid bilayer, and its dimensionality is wide enough to translocate canonical B-form double-stranded DNA (dsDNA). To broaden the analytical application of this γ-HL protein channel, we explore its ability to sample and distinguish nucleic acid conformational polymorphisms, such as A-, B-, or Z-form helixes, with single-molecule resolution. The canonical B-form structure and the slimmest left-handed Z-form DNA duplexes translocate through the γ-HL nanopore; in contrast, the A-form DNA-RNA hybrid duplex with a wider diameter is unable to translocate through the protein channel until unzipping into its two single-stranded components. The current signatures reveal multiple interactions between the nucleic acids and the γ-HL nanopore. Our results introduce the γ-HL protein channel (HlgB-HlgC) as an analytical tool for nucleic acid analysis at the single-molecule level. Our results demonstrate that this protein nanopore can be used to investigate the conformational heterogeneity and structural transitions of duplex nucleic acids.