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NORTHWESTERN UNIVERSITY the Ionic Environment and Solution NORTHWESTERN UNIVERSITY The Ionic Environment and Solution Interactions of Protein Spherical Nucleic Acids Probed by In-situ X-ray Scattering A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS for the degree DOCTOR OF PHILOSOPHY Field of Applied Physics By Kurinji Krishnamoorthy EVANSTON, ILLINOIS December 2018 2 © Copyright by Kurinji Krishnamoorthy. 2018 All Rights Reserved 3 ABSTRACT The Ionic Environment and Solution Interactions of Protein Spherical Nucleic Acids Probed by In-situ X-ray Scattering Kurinji Krishnamoorthy Electrostatic interactions mediated by ionic environments play a central role in physical processes across materials science, chemistry and biology. Key biological phenomena, such as the condensation and packaging of DNA, ion transport across cellular membranes and the enzymatic action of proteins, rely on the complex interplay between nanoscale electrostatic, osmotic and entropic forces. A consideration of such interactions is especially relevant to synthetic bioconjugates, which harness the powerful properties of molecules, such as proteins and nucleic acids to realize applications in materials assembly and therapeutic medicine. Spherical nucleic acids (SNAs) defined as a dense three-dimensional arrangement of oligonucleotides on the surface of a particle core are one such striking example of a bioconjugate whose collective properties are distinct from those of its nucleic acid components. For instance, the DNA shell and associated counterionic cloud on an Au nanoparticle SNA results in highly programmable assembly behavior, cooperative hybridization thermodynamics, efficient internalization across more than 200 different cell types and an enhanced resistance to enzymatic degradation in comparison to linear DNA. While the properties and assembly behavior of spherical nucleic acids are well characterized, the nanoscale structure and role of the counterionic cloud surrounding such constructs is poorly understood. Here, we address this challenge in the context of a spherical nucleic acid composed of a functional protein core using in-situ solution x-ray scattering techniques. Our approach provides 4 fundamental insights into the ionic environment around a protein spherical nucleic acid (Pro-SNA) and its influence on the resistance of Pro-SNAs to enzymatic degradation. 5 ACKNOWLEDGEMENTS I would first like to thank my advisors, Professor Michael Bedzyk and Professor Chad Mirkin for the incredible opportunity to work with two uniquely diverse research groups on some truly interdisciplinary projects spanning materials science, chemistry and x-ray physics. They have always been extremely supportive and have taught me how to be a better scientist and communicator. I am grateful for their unwavering support and find myself incredibly fortunate to have had the opportunity to work with two pioneers in their respective fields. Secondly, I would like to thank the other members of my committee, Professor Olvera de la Cruz and Professor Pulak Dutta for their invaluable advice, collaboration and helpful discussions. Collaborating with Professor Olvera de la Cruz and members of her group has been an amazing learning experience and has allowed me to gain insight into the theoretical considerations underlying our research. I would also like to thank Dr. Sumit Kewalramani for his mentorship and guidance. He has always been extremely helpful and knowledgeable about x-ray theory, techniques and modelling and is always willing to help in problem-solving and brainstorming new approaches. I would also like to acknowledge my funding sources the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, DOE-BES, under award number DE-SC0018093, MURI: BioProgrammable One-, Two-, and Three-Dimensional Materials under Grant Number: FA9550-11-1-0275 and a fellowship from the Center of Computation and Theory of Soft Materials at Northwestern University. Without this financial support, my graduate research work would not have been possible. I also thank the beamline scientists at the Advanced Photon Source, for their help in acquiring X-ray data, including Dr. Steven Weigand, and Dr. Soenke Seifert. 6 I would like to thank my collaborators in the Mirkin and Olvera de la Cruz groups – Dr. Jeffrey Brodin for teaching me how to synthesize and characterize protein spherical nucleic acids, Dr. Kyle Hoffmann and Annaliese Ehlen for their useful discussions regarding DFT and MD simulations. I would also like to thank my mentor Dr. Liane Moreau who was always incredibly supportive and taught me how to design and execute a good experiment. I’m grateful for the opportunity to have met, talked to and worked with so many intelligent and talented researchers across the Materials Science, Chemistry and Physics departments and truly cherish my time immersed in the interdisciplinary research environment at Northwestern. I thank all the members of the Mirkin and Bedzyk group for their support and friendship – Stephanie Moffitt, Bor-Rong Chen, Gavin Campbell, Changrui Gao, Katherine Harmon, Elise Goldfine, Anusheela Das, KVLV Narayanachari, Li Zeng, Xiao Chen and Guennadi Evmenenko. I would also like to thank my friends and family for their steadfast support and companionship. I’d like to thank my parents who have always supported everything I have wanted to do and have been a constant source of encouragement and inspiration. I would like to thank my friends Anjali, Kaushik, Madhu, Riju and Sonali and my boyfriend Nikhil for being my support system and in many ways my family away from home. I would also like to thank the members of the Evanston Animal Shelter for giving me the opportunity work with some amazing people and animals and meet my cat, Mia who has been a loyal companion throughout my graduate career. 7 Table of Contents ABSTRACT ................................................................................................................................... 3 Chapter 1: Introduction ................................................................................................................. 20 Chapter 2: Methods ....................................................................................................................... 25 2.1. Synthesis of Protein Spherical Nucleic Acids ................................................................ 25 2.2. Small Angle X-ray Scattering ....................................................................................... 27 2.2.1. Anomalous Small Angle X-ray Scattering .......................................................... 31 2.3. SDS Polyacrylamide Gel Electrophoresis ..................................................................... 34 2.4. Dynamic Light Scattering .............................................................................................. 36 2.5. Circular Dichroism Spectroscopy .................................................................................. 38 Chapter 3: Defining the Structure of a Protein Spherical Nucleic Acid Conjugate and its Counterionic cloud ........................................................................................................................ 41 Abstract ................................................................................................................................. 41 Introduction ........................................................................................................................... 42 Materials and Methods .......................................................................................................... 46 1. Sample preparation ................................................................................................... 46 2. X-ray Measurements ................................................................................................. 48 3. Density Functional Theory (DFT) ............................................................................ 49 Results and Discussion ......................................................................................................... 50 Summary and Conclusions ................................................................................................... 62 Proposed Future Work .......................................................................................................... 64 1. Counterionic Distribution Profiles at physiological salt concentrations ................... 64 8 2. Counterion Distribution surrounding Pro-SNA superlattices ................................... 65 Chapter 4: The Enzymatic Degradation of Protein Spherical Nucleic Acids probed by In-Situ X- ray Scattering ................................................................................................................................ 67 Abstract ................................................................................................................................. 67 Introduction ........................................................................................................................... 68 Materials and Methods .......................................................................................................... 72 1. Synthesis of Pro-SNAs ............................................................................................. 72 2. X-ray Scattering Measurements ................................................................................ 73 3. Gel Electrophoresis Measurements (SDS PAGE) ...................................................
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