Unzipping kinetics of duplex DNA containing lesions in a nanopore and its application in monitoring an enzyme reaction

The dissertation presents biophysical studies of duplex DNA during unzipping in a protein ion channel and its application in the fast readout of enzyme activity. Chapter 1 reviews a variety of approaches to enhance the resolution of nucleotide characterization using nanopore methodology, providing insights into future experimental design. This chapter also overviews the work carried out at University of Utah concerning the characterization of DNA damage using nanopore methods. Chapter 2 examines the unzipping kinetics of lesion-containing duplexes in the protein channel a-hemolysin (a-HL). The voltage-driven unzipping of individual duplex DNA molecules was investigated by pulling the tail of duplex into the a-HL and monitoring the temporal blockage to the ion flow through the channel. The unzipping occurs as a first-order reaction or sequential first-order reactions as determined from the time duration histograms of the blockages. Chapter 3 extends the unzipping studies of duplex to a larger pool of lesions. As a more destabilizing lesion is introduced into the duplex, the process of strand dissociation grows faster and evolves from a first-order reaction to two sequential first-order reactions. The kinetic stability of duplexes was interpreted in terms of the lesion-induced distortion of duplex backbone as the major factor and the number of hydrogen bonds in the modified base pair as the minor factor. Chapter 4 provides an application of duplex unzipping in the measurement of enzyme activity. Specifically, the conversion of uracil to an abasic site by uracil DNA glycosylase (UDG) was monitored using ion-channel recordings based on the difference in blockage current. The single-nucleotide discrimination during UDG digestion was performed in the duplex context at the latch region of the a-HL. The data suggest that the protein latch is a new sensing zone in a-HL, specifically useful for duplex analysis.