Development of Nanopore-Based Analytical Tools for Single-Molecule Analysis of Biological Molecules

Single-molecule analysis has been matured into powerful and popular tools for study the heterogeneous behavior of biological molecules, which is often difficult or impossible to observe using bulk detection methods. Nanopore sensing has become a powerful tool for single-molecule analysis of a broad spectrum of analytes ranging from small molecules to proteins and nucleic acids. Our previous studies have shown that due to its highly dynamic and flexible loop structure, outer membrane protein G (OmpG)-based nanopore possesses a unique feature to resolve highly homologous protein in serum. Our first generation of OmpG nanopore sensors contains a ligand chemically tethered to loop 6 of OmpG. However, this approach is limited by the incomplete labeling efficiency and the requirement for a known ligand. In this work, I have developed the next generation of OmpG nanopore sensor by introducing the peptide recognition sequence into the loops. The second-generation of OmpG design retains its ability to discriminate protein homologs in a complex mixture of bacterial cell lysate. Furthermore, I have initiated the construction of a bacterial surface display library for high-throughput screening of OmpG nanopore constructs. Synthetic DNA holds promise as a data storage medium with ultrahigh information density capabilities. Nanopore sequencing offers a portable, fast, inexpensive, and real-time decoding strategy for the molecular storage system. MspA is a rigid nanopore that has been used for DNA sequencing. In this thesis, we have assessed the ability of MspA to discriminate modified unnatural DNA nucleotides, which could expand the four nature nucleotide alphabet. Our results reveal that MspA can distinguish all four natural and five modified DNA nucleotides highlighting the importance of MspA as the decoding read-head in the future of DNA data storage. Last, we have studied fundamental questions about the role of outer membrane porins in bacterial conjugation, one of the main mechanisms by which bacteria acquire antibiotic resistance. Our results provide evidence for the role of OmpK36 as the DNA conduit in the conjugal transfer of Klebsiella pneumoniae carbapenem resistance plasmid. It was the first revelation that single-stranded DNA can be translocated through a trimeric porin like OmpK36