Unfolding of Nanoconfined Circular DNA

Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.Circular DNA is of interest since it is found in many biologically relevant contexts, such as bacterial plasmids, viruses and eukaryotic mitochondrial DNA. Furthermore, the circular topology forces two strands in close proximity to each other in nanochannel, which changes the polymer physics compared to linear DNA. Circular DNA is difficult to study with traditional single molecule techniques because such techniques generally require the attachment of handles to the, but is readily accessed using nanofluidics.Circular DNA has less entropy and higher conformational free energy than in its unfolded configuration. Therefore, as a double-strand break occurs and circular DNA opens up, it unfolds to its linear configuration inside the nanochannel. This study compares the static properties of confined linear and circular DNA as well as investigates the dynamics of the transition from circular to linear DNA as a double-strand break occurs.We observe that the difference in extension between the circular and linear configurations depends on the degree of confinement, which we confirm with theoretical predictions. Our data for unfolding of the circular DNA to the linear configuration suggests that hydrodynamic friction between the DNA and the solvent is the main rate-determining factor but that DNA-DNA contacts are also important. Finally, by staining the DNA inhomogeneously, we can follow the local dynamics of the DNA as the folding occurs and conclude, for example, how the two different strands move relative to each other during the unfolding process. We are thus able to study the dynamics of confined DNA with unprecedented resolution and obtain completely new information about confined polymers.