Measuring and controlling the position of DNA in a nanopore to explore DNA-polymerase interactions at the single molecule level

Biological nanopores have emerged as a powerful tool for examining the kinetics of DNA and DNA-binding proteins at the single molecule level. A biological nanopore such as alpha-hemolysin is a protein channel inserted into a lipid bilayer separating two volumes of an ionic solution. A voltage is then applied across the bilayer enabling an ionic current to flow through the nanopore. The negatively charged backbone of DNA in solution can be drawn toward and captured in the nanopore by the applied trans-membrane voltage. The structure of the pore will allow the passage of single-stranded DNA but not double-stranded DNA. Nanopore capture of a DNA molecule causes a large decrease in the ionic current. For certain proteins, protein-bound DNA has a detectably different current amplitude than unbound DNA: a difference that can be used to detect bound DNA and subsequent protein dissociation from the DNA captured in the pore. The nanopore is thus an ideal instrument to study the kinetics of duplex DNA and enzymes that bind to these DNA substrates.;The first part of my thesis explores DNA-DNA polymerase complexes captured in the nanopore. To gain a detailed understanding of the interaction of a polymerase with its primer/template substrate, the position of polymerase bound DNA within the nanopore needs to be accurately determined. Using abasic residues in DNA templates, this work demonstrates that the position of polymerase-bound DNA in a nanopore can be measured at single nucleotide (5A) resolution.;Secondly, my thesis examines the kinetics of DNA hybridization in proximity to a nanopore. DNA hybridization is explored by allowing an oligonucleotide to bind to a complimentary portion of the DNA template that is captured and threaded through the nanopore. By varying the length of the template and the duration that it is available for binding, hybridization kinetics are measured and modeled to quantitatively understand this process near the nanopore.;A unique aspect of the nanopore is that large amounts of individual molecules can be measured over the course of an experiment. This necessitated the development of software to efficiently analyze the results. This dissertation describes the development and implementation of custom software that accurately and quickly analyzes data from nanopore experiments.