Kinetics of polymer cyclization reaction and novel covalent dna cross-linking assays

Reza Afra, Purdue University

Abstract

In this dissertation I first do an extensive review of polymer cyclization kinetics. Different theories of polymer cyclization kinetics, their assumptions and their predictions are presented along with the predictions of computer simulations. In addition, the experimental results for synthetic and biological polymers are summarized. Secondly, from our Brownian dynamics simulations of the worm-like chain we discovered that the polymer cyclization kinetics cannot be adequately described by transition-state-like theories that reduce the high dimensional kinetics to a one dimensional diffusion along a reaction coordinate. It is common in Brownian dynamics simulations of WLC to discretize time step. In our simulations, however, we recovered the continuum limit for time by extrapolating the time steps to zero and found that finite time steps lead to erroneous results for cyclization kinetics. This is the only work to date that shows the inadequacy of 1D diffusion-reaction models in capturing the kinetics of polymer cyclization. Lastly, we developed novel assays for covalently crosslinking DNA. To this end, we screened different crosslinking methods and found that Iodine-mediated disulfide bonding and copper-free azide-alkyne cycloaddition are the most viable paths to ligase-free DNA crosslinking. We developed methodologies for labeling and purification of dsDNA with those reactive moieties and carried out experiments to test the yield of these chemistries at low DNA concentrations. Our results establish a method for crosslinking long linear dsDNA through their ends.

Degree

Ph.D.

Advisors

Todd, Purdue University.

Subject Area

Biophysics

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