School of Physics Thesis Dissertation Defense

Alexander W. Cook
Advisor: Dr. Harold Kim, School of Physics, Georgia Institute of Technology

Single-molecule kinetics of DNA branch migration, hybridization, and dehybridization
Date: Thursday April 24, 2025
Time: 12:30 p.m.
Location: Howey physics building N201/N202

Zoom link: https://gatech.zoom.us/j/96638489661?pwd=YFnV64F2BdpViTdffuPb1slDpGVfiL.1
Meeting ID: 966 3848 9661
Passcode: 833595

Committee members:
Dr. James Gumbart, School of Physics, Georgia Institute of Technology
Dr. Kurt Wiesenfeld, School of Physics, Georgia Institute of Technology
Dr. Brian Hammer, School of Biological Sciences, Georgia Institute of Technology
Dr. Yonggang Ke, Coulter Department of Biomedical Engineering, Georgia Institute of Technology

Abstract:
Strands of deoxyribonucleic acid (DNA) possess the remarkable ability to bind to complementary molecules in a specific, predictable fashion through a mechanism known as base pairing. Two strands come together to form a single duplex through a process called hybridization, while the reverse process, dehybridization, splits a single duplex into two strands. Branch migration is a third process that combines hybridization and dehybridization as two strands compete for base pairs with a third to which they are both complementary. This dissertation presents the results of single-molecule experiments designed to study the kinetics of these three processes. 

The first half of the dissertation is devoted to a first passage time study of branch migration within toehold mediated strand displacement (TMSD). We show that branch migration times are heavily sequence dependent, with displacement times varying by more than an order of magnitude. We show that complementary invasion systems, with each strand replaced by its reverse complement, display different kinetics, as do systems with RNA invaders. We demonstrate that an invader overhang which does not participate in base pairing slows invasion kinetics. We also build a model of branch migration kinetics to explain these observations, adopting a nonuniform, asymmetric random walk approach.

The second half of the talk presents the effect that DNA duplexes have on the hybridization and dehybridization kinetics of nonlocal duplexes. We find that hybridization and dehybridization are both suppressed in the presence of a flanking duplex motif separated by a single-stranded gap. We present coarse-grained simulations which show that flanking duplexes increase the extension of single-stranded DNA through a combination of steric and electrostatic interactions. We also present all-atom simulations which suggest the stabilizing effect of flanking duplexes is due to stacking interactions mediated by the single-stranded gap. Finally,  we show that surface treatments and linker chemistry can affect hybridization kinetics.