All ETDs from UAB

Advisory Committee Chair

Michael Niederweis

Advisory Committee Members

Kevin L Kirk

Peter E Prevelige

David A Schneider

Charles L Turnbough

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Nanopore sequencing is a novel and promising DNA sequencing method. This single molecule technique is capable of long reads, retains epigenetic information, and is inexpensive and fast. In this method ionic current is measured while single-stranded DNA is electrophoretically translocated through a nanometer-scale pore. Each passing nucleotide blocks current with characteristic amplitude and duration which are used to identify DNA sequence. MspA is the primary porin of M. smegmatis and mediates the diffusion of small, hydrophilic nutrients across the outer membrane. MspA is an octameric, channel-forming protein and represents the founding member of a new class of mycobacterial outer membrane proteins. Its short and narrow constriction zone makes MspA ideal for nanopore DNA sequencing. However, wild-type MspA does not translocate DNA. In this thesis, we identified key positions in MspA involved in translocation of DNA and engineered MspA mutants to translocate single-stranded DNA. In a breakthrough study, these engineered MspA pores enabled DNA sequencing and the detection of methylated nucleotides. Next, we improved the purification of MspA mutants by constructing a porin deletion mutant of M. smegmatis lacking all msp genes. Pores purified from this strain had a smaller single-channel conductance distribution indicating the absence of background Msp contaminants. Due to the octameric symmetry of MspA, precise control of the chemical properties of the pore with atomic precision is challenging. Therefore, we constructed a single-chain mspA gene in which all eight subunits are connected together by regions encoding a peptide linker. We showed that linking MspA subunits does not change the channel-forming properties of MspA. We identified MspA’s region responsible for the reconstitution into lipid bilayers and improved the membrane insertion of single-chain MspA. Single-chain MspA enables to design pores with a single mutation and thereby enables a better control of the pore properties. In conclusion, we provided in this thesis the basis for using MspA as a pore for DNA sequencing and showed that single-chain MspA provides a highly efficient platform for fine-tuning DNA translocation.

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