| Description |
Nanopores provide a unique platform for single-molecule detection with broad biological applications, such as DNA sequencing, protein folding/unfolding detection, protein-DNA/peptide interactions, and enzyme kinetics measurement. In this dissertation research, the ability to discriminate base modifications including DNA backbone damage in double-stranded DNA (dsDNA) using biological nanopores was demonstrated. Chapter 1 reviews the applications of both biological and artificial nanopores in detecting ssDNA and dsDNA. The latch sensing zone of α-hemolysin (α-HL) nanopore has been used to demonstrate a single base modification in the double-stranded DNA (dsDNA). Chapter 2 describes that phosphodiester backbone damage in dsDNA can also be differentiated from the intact duplex, with a significant increase in both the blockade current and noise level. Furthermore, this baseline-resolved differentiation between the nicked and intact duplexes allows the real-time monitoring of the phosphodiester bond formation by the T3-DNA ligase. Due to the pore geometry of α-HL, dsDNA with a wider diameter than the protein constriction zone is trapped in the vestibule and denatured into its corresponding complementary strands. Thus, the search for a protein nanopore to translocate dsDNA with the ability to differentiate modified bases in the dsDNA is still ongoing. The γ-hemolysin (γ-HL) protein produced by Staphylococcus aureus is able to assemble into an octamer nanopore with a ~2.3 nm diameter β-barrel, with the potential to be a distinct biological sensor for dsDNA detection. The dsDNA-nanopore interactions depend strongly on the nucleic acid conformation, A-, B- or Z-form (Chapter 3-4). The canonical B-form structure and the slimmer left-handed Z-form DNA duplexes translocate through the γ-HL protein channel as a duplex; in contrast, the wider A-form DNA/RNA hybrid duplex unzips during translocation through the γ-HL nanopore. Exploiting the distinctive geometry of γ-HL, the resulting current signatures reveal multiple mechanisms for the unzipping, translocation, and conformational changes of nucleic acids duplexes in the γ-HL protein channel. The current modulation before the B-form dsDNA translocation process provides a unique signature to distinguish the guanine-to-inosine substitutions from the wild-type duplex. These results indicate the γ-HL channel has a great potential to investigate modified base detection, nucleic acid conformational heterogeneity, and gene delivery. |
