Thesis Dissertation Defense

Abstract: DNA duplex separation and formation underlie our most fundamental genetic processes. One of the most powerful tools for exploring these binding and unbinding reactions is single-molecule force spectroscopy. This method applies tension to a single DNA strand and observes the change in its extension or kinetics with a microscope. However, these experiments typically study longer DNA molecules (>10 bp) subject to higher forces (> 10 bp); hence, the behavior of short DNA subject to weak forces is not well understood. In particular, it is not clear whether statistical chain models ordinarily used to explain force spectroscopy data are applicable at these small scales. To remedy this, we focused solely on short DNA duplexes (< 10 bp) subject to very weak forces (< 7 piconewtons). For this purpose, we used two tools: an experimental technique called single-molecule fluorescence resonance energy transfer (smFRET), and the coarse-grained DNA simulation code oxDNA. The experiments implemented a simple, high-throughput DNA assay, dubbed “DNA bows”, which exploit the bending rigidity of DNA to exert very weak forces on short DNA strands. With this method, we demonstrate that weak force accelerates the hybridization and dehybridization of short oligonucleotides from 2-6 piconewtons, contradicting the predictions of simple chain models. We next used oxDNA to investigate how the extension of short DNA changes with force throughout its binding and unbinding transitions. Regardless of force, we find that the transition state of an oligoduplex is at least as extended as its bound state, in agreement with our experiments. We also find that extension is a poor reaction coordinate at 3 piconewtons and below. These results establish the nature of DNA duplex transitions at the force and length scales relevant to DNA biology as well as emerging DNA nanotechnologies.