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Design, Synthesis, and Characterization of Functional Click Nucleic DESIGN, SYNTHESIS, AND CHARACTERIZATION OF FUNCTIONAL CLICK NUCLEIC ACID POLYMERS AND CONJUGATES FOR BIOLOGICAL APPLICATIONS By ALEX J. ANDERSON B.S., Vanderbilt University, 2015 M.S., University of Colorado, 2017 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemical and Biological Engineering 2020 Thesis committee: Dr. Christopher N. Bowman, Chair Dr. Stephanie J. Bryant Dr. Virginia L. Ferguson Dr. Jennifer N. Cha Dr. Frank Vernerey Abstract Anderson, Alex J. (PhD. Chemical and Biological Engineering) Design, Synthesis, and Characterization of Functional Click Nucleic Acid Polymers and Conjugates for Biological Applications Thesis directed by Christopher N. Bowman and Stephanie J. Bryant Oligonucleotides are a powerful class of biopolymer which, through Watson-and-Crick hydrogen bonding, are capable of directing the self-assembly of materials as well as recognizing and binding to very specific targets. These features have made oligonucleotides an attractive tool for uses in materials science and biotechnology. However, there are several drawbacks to the implementation of oligonucleotides with a natural backbone (i.e. DNA or RNA) including susceptibility to degradation and a limited scale of production. Click Nucleic Acids (CNAs) represent a new class of oligonucleotide that was designed to alleviate these issues. The distinguishing feature of CNAs is that they are polymerized via radical mediated thiol-ene click chemistry, which vastly increases the speed and scale of oligonucleotide synthesis. This thesis focuses on developing CNA polymers and conjugates and demonstrating the utility of CNAs as an alternative oligonucleotide for various biological applications. Initially, linear polymers and copolymers were synthesized and CNA’s cytocompatibility and ability to interact with cellular components was evaluated. Specifically, it was found that linear PEG-CNA conjugates were taken up by cells quickly through an apparently passive mechanism and did not exhibit significant cytotoxicity. In addition, the ability of CNA homopolymers to bind to mRNA was demonstrated by evaluating its use as an mRNA isolation technique. Taking advantage of the ii inherent insolubility of CNA, functional mRNA was effectively isolated from total RNA extracts in yields comparable to commercially available products as determined by in vitro protein translation and RT-qPCR. The second part of this thesis details the synthesis of a branched CNA conjugate and its ability to form physically crosslinked networks upon the introduction of complementary ssDNA. These gels were found to be viscoelastic and completely thermoreversible, exhibiting a melting transition around 60°C. In all, microscale thermophoresis, circular dichroism spectroscopy, and rheology were all used to study the complex crosslinking interaction between CNA and DNA. Finally, higher order, CNA-microparticle conjugates were developed and their ability to load and delivery ssDNA was assessed. After confirming successful conjugation, CNA functionalized microparticles were found to load approximately 6 pmol ssDNA / mg microparticle and loading was sensitive to ssDNA length and sequence. Release of loaded ssDNA was determined to be temperature dependent, but stable to buffer pH. Further, phagocytosis of microparticles was observed via fluorescence microscopy and corroborated by biochemical analysis. In all, this thesis demonstrates the uses of CNA as a functional oligonucleotide for a variety of biological applications through the design of various conjugate architectures. iii To my Dad and Mom Dad, for inspiring me, every single day Mom, for the love and support you have always provided iv Acknowledgements This dissertation is the culmination of not only years of work, but also years of relationships with my mentors, colleagues, and friends. There is not a way to adequately thank all of the people who have helped me during this process, but I will do my best here. First, I would like to thank my advisors, Christopher Bowman and Stephanie Bryant. I could not have asked for a more supportive pair of mentors. I often got a lot of questions about being a co-advised student and I always say I got the best of both worlds. Your varied expertise challenged me to think about problems from both a materials science and biological perspective which I think is one of the most important skills I developed in graduate school. I thank you both for your guidance and mentorship which have prepared me to take the next steps in my career. I would like to thank my committee, Ginger Ferguson, Jennifer Cha, and Franck Vernerey for their valuable input throughout my years of research. This project got to where it is today through the help of you and your students. In addition, I want to thank all of my collaborators that have helped me on this project. In particular, I want to thank Heidi Culver, Ben Fairbanks, Jasmine Sinha, and Mingtao Chen for lending scientific advice and allowing me to vent when things went awry. This project was made possible through a National Science Foundation supported Materials Research Science and Engineering Center (DMR 1420736). Personally, I was funded by a US Department of Education Graduate Assistantship in Areas of National Need (GAANN) grant. I also would like to thank my undergraduate advisors for encouraging my interest in research and for providing me with opportunities for me to learn new skills and explore different areas of research. Dr. Guelcher and Dr. Duvall, thank you for your willingness to take on an inexperienced undergraduate student and for helping me find my way. v The best piece of advice I can give to new graduate students is to make sure you are surrounded by people you love and who love you. At the end of the day, these are the people who will be your support when things become stressful due to deadlines, important presentations, or more often failures. In my case, I am lucky that I have had such an incredible support system at CU-Boulder, and I would not trade my experience for the world. My labmates in both the Bowman and Bryant research groups are all wonderful people and I cannot thank them all enough for making lab a fun place to work. You all were an integral part to my experience here and made me feel like family from day one. I especially want to thank Elizabeth and Stanley for being awesome lab mentors and more importantly, great friends. I know I asked a lot of stupid questions, but I want to thank you for answering them earnestly and making me feel like I was a part of the lab. Margaret, thank you for being a great labmate, roommate, role model, and friend. When the goings got rough, you always provided a level-headed perspective and weren’t afraid to let me know when I was overreacting, which I sincerely appreciate. Parker also told me to thank Dorie for being his best friends. And Leila, thank you for being my best friend. Your dad was right, your grad school friends will be your friends for life, despite anything anyone says (including me). Oh, and Parker wanted me to thank Butler for being his other best friend. Finally, to all my family and friends everywhere, thank you for your constant love and support! vi Table of Contents Chapter 1 - Introduction .................................................................................................................. 1 1.1 Introduction ........................................................................................................... 1 1.2 Uses of DNA-Polymer Conjugates ....................................................................... 3 1.2.1 Linear Architectures ........................................................................................ 4 1.2.2 Branched Architectures ................................................................................... 5 1.2.3 Higher Order Architectures ............................................................................. 6 1.3 Development of XNA’s and their Polymer Conjugates ........................................ 7 1.3.1 Peptide Nucleic Acids ..................................................................................... 8 1.3.2 Locked Nucleic Acids ................................................................................... 11 1.3.3 Morpholino Nucleic Acids ............................................................................ 12 1.3.4 Other types of XNAs ..................................................................................... 14 1.4 Development of Click Nucleic Acids .................................................................. 15 1.5 Thesis overview ................................................................................................... 17 1.6 References ........................................................................................................... 18 Chapter 2 - Objectives and Scope ................................................................................................. 28 2.1 Aim 1: Identify interactions between linear CNA polymers and copolymers with cells cellular components .................................................................................... 29 2.1.1 Sub-aim 1.1 ................................................................................................... 29 2.1.2 Sub-aim 1.2 ................................................................................................... 30 2.2 Aim 2: Utilize
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