Dynamics of Knotted DNA Molecules Confined in Nanochannels

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Dynamics of Knotted DNA Molecules Confined in Nanochannels

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Knots are intriguing topological objects and ubiquitous in biopolymers such as DNA molecules. The occurrence of knots in DNA confounds the accuracy of genomics technologies, such as nanochannel-based genome mapping and nanopore sequencing, that require uniformly stretching the DNA molecules. Knots existing in vivo also influence biological processes, such as DNA replication, and hence leads to cellular malfunction. The control of DNA knots is, thus, significant for genomics technologies and cell survival, which require first understanding the fundamental properties of knotted DNA in a crowded environment. The aim of this thesis is to address fundamental questions related to knot transport in nanochannel-confined DNA molecules, particularly the knot diffusion mechanism, the effect of knots on DNA diffusion in nanochannels and the interactions between two knots. We first determined the knot diffusive behavior along DNA confined in nanochannels to distinguish between two predicted knot diffusion mechanisms, self-reptation and knot region breathing. With a recently developed nanofluidic "knot factory" device, we generated knots in DNA molecules efficiently. The experimental results of knot motion along DNA chains show that knots undergo subdiffusion, i.e. their mean-squared displacement grows sublinearly with time, which supports the knot diffusion mechanism of self-reptation. We then investigated the effects of knots on DNA center-of-mass diffusion in nanochannels, thus resolving the open question which of these competing effects, the shortening of DNA chains or the increased DNA-wall friction, dominates knotted DNA diffusion in nanochannels. To address this question, we measured the diffusivity of DNA molecules before and after knot formation via a combination of the nanofluidic knot factory device for knot generation and laser-induced fluorescence microscopy for DNA observation. The experimental results show that the presence of knots decreases the diffusivity of DNA chains confined in nanochannels. The reduced diffusivity indicates that the DNA-wall friction, rather than the shortening of the confined chain size, dominates the friction of knotted DNA in nanochannels. Our previous work focused on the dynamical properties of single knots. Long DNA molecules are susceptible to form multiple knots in the chains. In the third research project, we investigated the interactions between two knots in nanochannel-confined DNA by analyzing the motion of the two knots along DNA chains. The free energy profiles of knot-knot interactions show that the separated knot state is more stable than the intertwined knot state, with dynamics in the separated knot state that are consistent with independent diffusion of the two knots. The thesis work provides deep insights into the dynamical properties of DNA knots under nanochannel confinement. We hope such fundamental knowledge gained in this dissertation could prescribe avenues for suppression and removal of knots under nanofluidic systems and crowded environments, thereby improving the genomic technologies and controlling knots in living cells.


University of Minnesota Ph.D. dissertation. 2022. Major: Chemical Engineering. Advisor: Kevin Dorfman. 1 computer file (PDF); 152 pages.

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Ma, Zixue. (2022). Dynamics of Knotted DNA Molecules Confined in Nanochannels. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/241430.

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