Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics

Abstract

Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of DNA from the circular to linear configuration as a light-induced double strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to which extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.

Stochastic unfolding of nanoconfined DNA: Experiments, model and Bayesian analysis

“…[44,45] We compare the predictions of our model to new experimental data. The experiments reported here use the same method as [48], but here the DNA is much longer which makes the unfolding slower and easier to analyze in detail. We label the DNA with the fluorescent dye YOYO-1 and exploit the fact that this dye forms reactive oxygen species in its excited state.…”

Wall depletion length of a channel-confined polymer

“…DNA has played a key role in the experimental tests of theories of a polymer confined to a slit [1][2][3][4][5][6][7][8][9][10][11][12][13][14] or a channel [15][16][17][18][19][20][21][22][23][24][25][26][27], in particular the models by de Gennes and coworkers for weak confinement [28,29] and Odijk for strong confinement [30,31]. The key results of this body of work have been summarized in several recent reviews [32][33][34].…”

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

“…To confirm the presence of a plasmid of a certain size, we intentionally did not run the reaction to completion, so that some plasmids remained circular. This is important because we can undisputedly confirm that plasmids of a certain size are present in the sample if we detect intact circular plasmids of the corresponding size1619. Only linear fragments of a size corresponding to that of a circular plasmid detected are used in the analysis below.…”

“…Using the extension of imaged λ-DNA molecules, a reference value of number of basepairs per pixel for each separate experiment was obtained, allowing for plasmid sizing. Intact plasmids are circular by nature, making it possible to separate linear fragments from circular when extended in the nanochannels due to the higher intensity of emitted light from the double folded plasmids19. Plasmids in their native circular form, which will be present even if the plasmid is targeted with Cas9 since the reaction is not run to completion, were used to calculate the sizes of plasmids present in the isolates.…”

“…Plasmids in their native circular form, which will be present even if the plasmid is targeted with Cas9 since the reaction is not run to completion, were used to calculate the sizes of plasmids present in the isolates. To convert the sizes of circular plasmids to the linear form we use a previously reported1419 conversion factor of 1.8. For each detected plasmid size all linear DNA fragments +/− 20% in size were then merged into a cluster.…”