Date of Award

Winter 2025

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

First Advisor

Nathan J Oldenhuis

Second Advisor

Glen Miller

Third Advisor

Roy Planalp

Abstract

Double-stranded DNA (dsDNA) exhibits many unique sequence-independent properties that remain underutilized in materials science. The exceptional length, solubility, biocompatibility, imageability, molecular recognition capabilities, and topological control of DNA makes it an ideal candidate for the creation of bespoke biomaterials and investigation of fundamental relationships in polymer physics. Unfortunately, translation of DNA-based materials from the microscale to macroscale has been hindered by source constraints and limitations in chemical modification of biologically derived DNA. This work seeks to expand the utility of dsDNA from the benchtop to real world applications. Establishing dsDNA as a commodity polymer will aid in the development of customized materials and topologically complex polymeric systems.To address the source limitation, we engineered a robust, scalable platform for the gram-scale production of plasmid DNA (pDNA) via fed-batch fermentation and a modified alkaline lysis protocol coupled with anion exchange chromatography for purification. This process yields highly concentrated DNA solutions (up to 116 mg/mL, ~70c*) suitable for bulk rheological investigations and now serves as the foundation for several projects in our group. The isolated pDNA can be enzymatically linearized or used in native supercoiled (SC)/open circle (OC) isoforms which enabled us to systematically study topological effects on macromolecular behavior. Using this platform, we applied bulk rheology to linear and SC/OC DNA solutions across various concentrations and lengths, generating a time-concentration superposition that spans 12 decades of frequency, which is the most extensive to date for DNA-based materials. As a result, we observed distinct viscoelastic properties, entanglement dynamics, and nonlinear flow behavior in both linear and ring polymer architectures. These results offer new insights into the reptation and relaxation dynamics of topologically complex polymers and establish dsDNA as a high-fidelity, unimolecular probe for soft matter physics. To further expand the utility of dsDNA as a commodity polymer, we next focused on chemical functionalization of biologically derived DNA. Crucially, such modifications must preserve dsDNA’s structural integrity to retain its unique properties within the material. Common acrylate and epoxide cross-linkers only react with single-stranded (ssDNA), requiring denaturation prior to material formation, causing a loss of emergent properties specific to dsDNA. To overcome this, we designed a series of monofunctionalized polyethylene glycol monomethyl ether (mPEGs) outfitted with nitrogen mustard derivatives. Nitrogen mustards are a class of DNA alkylators that contain chloroethylamine (CEA) moieties, which are known to react with the exposed N7 position on guanine, facilitating modification without compromising base pairing. To demonstrate the feasibility of this approach, we made topologically defined linear and circular DNA bottlebrush polymers (BBPs). Utilizing a graft-to approach, cyclic and linear DNA backbones were chemically modified with PEG monomethyl ether (mPEG) functionalized with CEAs (mPEGCEA) to generate DNA BBPs. Using this platform, we demonstrated that topology does impact material behavior. Linear BBPs showed reduced viscoelastic moduli, consistent with decreased entanglements, while cyclic BBPs exhibited enhanced moduli, suggesting the formation of more interchain interactions. This PEGylation method also conferred nuclease resistance, underscoring its potential for creating stable nucleic acid-based materials. Together, the work in this dissertation establishes pDNA as a viable platform for the broad implementation of dsDNA as a commodity polymer. With scalable production methods and chemistries that preserve structural integrity, future efforts will focus on refining these systems to create improved biomaterials. Planned work includes the synthesis of DNA-alkylating ATRP agents for graft-from BBP synthesis and the separation of SC and OC isoforms to further investigate structure – property relationships.

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