Leveling Up DNA Hybridization Affinity and Specificity Using Heteromultivalent DNA-Functionalized Particles Public

Abstract

Beyond the essential role that DNA hybridization serves in biological processes, it has also been demonstrated to be a powerful tool that enables many biological investigations and transformative applications in diagnostics, therapeutics, and nanotechnology. Despite the success of DNA hybridization-based applications, further enhancements in binding affinity and specificity are obstructed by several fundamental constraints. To bypass these obstacles, multivalent DNA hybridization has emerged as a promising strategy due to its ability to yield highly avid and specific binding. Numerous examples of multivalent DNA hybridization have been exhibited in recent years and have found significant utility in fields ranging from nanoparticle-based sensing to fundamental immunology. This dissertation begins by describing the fundamental parameters that influence multivalent DNA hybridization and highlighting several areas that have greatly benefited from the application of this concept. Following this guide to understanding and applying multivalent DNA hybridization, this dissertation presents two fundamental explorations into designing DNA-functionalized particles capable of high avidity and specificity binding to biologically relevant DNA targets using heteromultivalent DNA hybridization. Finally, essential steps towards further application of multivalent DNA hybridization in nanotechnology, sensing, and gene regulation are outlined.

Table of Contents

Table of Contents

 

Chapter 1. Overview of the Fundamental Concepts and Applications of Multivalent DNA Hybridization…………………………………………………………………………………….1

1.1. Introduction…………………………………………………………………………...2

1.2. Fundamentals of Multivalent DNA Hybridization…………………………………...5

1.2.1. How Does Multivalency Enhance Binding Affinity and Specificity?...........5

1.2.2. Defining Homomultivalent and Heteromultivalent DNA Hybridization…..7

1.2.3. The Spatial Arrangement of Oligonucleotides Significantly Impacts Multivalent Hybridization…………………………………………………………9

1.2.4. The Value of n Dictates Binding Valency of Multivalent Hybridization Interactions……………………………………………………………………….13

1.2.5. Individual Oligonucleotide Length and Monovalent Binding Affinity Impacts Binding Avidity and Selectivity………………………………………...16

1.2.6. Linkers and Spacers Increase Reach and Provide Flexibility to Multivalent Hybridization Interactions……………………………………………………….19

1.2.7. Anchor Orientation and Spacer Length Control the Binding Geometry of Multivalent Hybridization………………………………………………………..23

1.2.8. Quaternary Level and Higher Multivalent DNA Hybridization…………..26

1.3. Applications of Multivalent DNA Hybridization…………………………………...28

1.3.1. DNA-Based Artificial Motors……………………………………………..28

1.3.2. Nucleic Acid Sensing……………………………………………………...31

1.3.3. Tools to Study Biology……………………………………………………33

1.4. Aims and Scope of the Dissertation…………………………………………………36

1.5. References…………………………………………………………………………...38

Chapter 2. Engineering DNA-Functionalized Nanostructures to Bind Nucleic Acid Targets Heteromultivalently with Enhanced Avidity………………………………………………….42

2.1. Introduction………………………………………………………………………….43

2.2. Results……………………………………………………………………………….46

2.2.1. Modeling Binding of Random and Patterned HeteroMV SNAs………….46

2.2.2. Design and Melting Curve Analysis for Random HeteroMV SNAs……..48

2.2.3. Thermodynamics and Affinity of Random HeteroMV SNA Binding……50

2.2.4. Development and Characterization of Patterned HeteroMV SNAs………52

2.2.5. Impact of Spatial Patterning on HeteroMV SNA Binding………………..54

2.3. Discussion…………………………………………………………………………...55

2.4. Materials and Methods………………………………………………………………58

2.4.1. Oligonucleotides…………………………………………………………..58

2.4.2. Reagents…………………………………….……………………………..59

2.4.3. Consumables………………………………………………………………60

2.4.4. Equipment…………………………………………………………………60

2.4.5. Modeling…………………………………………………………………..60

2.4.6. Synthesis of Gold Nanoparticles…………………………………………..64

2.4.7. Random HeteroMV SNA Synthesis………………………………………64

2.4.8. Determining Number of Oligos/AuNP……………………………………65

2.4.9. Melting Curve Measurement for Random HeteroMV SNAs……………..66

2.4.10. Cy5 Conjugation to Target Strands………………………………………67

2.4.11. van’t Hoff Binding Affinity Measurement………………………………67

2.4.12. Native PAGE…………………………………………………………….68

2.4.13. Patterned and Mispatterned SNA Synthesis……………………………..69

2.4.14. Determining Number of Templates/Patterned SNA……………………..70

2.4.15. Melting Curve Measurement for Patterned SNAs……………………….70

2.5. Appendix…………………………………………………………………………….72

2.6. References…………………………………………………………………………...91

Chapter 3. Heteromultivalency Enables Optimization of the Specificity and Cooperativity of DNA Hybridization…………………………………………………………………………..94

3.1. Introduction………………………………………………………………………….95

3.2. Results……………………………………………………………………………….98

3.2.1. Modeling the Specificity and Cooperativity of HeteroMV Hybridization..98

3.2.2. Measuring the Specificity and Cooperativity of HeteroMV Hybridization……………………………………………………………...……101

3.2.3. Determining the Impact of Spacer Length on HeteroMV Hybridization Specificity and Cooperativity…………………………………………………..104

3.2.4. Determining the Impact of Binding Orientation on HeteroMV Hybridization Specificity and Cooperativity…………………………………...106

3.2.5. Detecting the Cis/Trans Relationship of Two Mutations Using HeteroMV Hybridization…………………………………………………………………...108

3.2.6. Distinguishing Different Strains of SARS-CoV-2 Using HeteroMV Hybridization…………………………………………………………………...111

3.3. Discussion………………………………………………………………………….113

3.4. Materials and Methods……………………………………………………………..117

3.4.1. Oligonucleotides…………………………………………………………117

3.4.2. Reagents………………………………………………………………….118

3.4.3. Consumables……………………………………………………………..119

3.4.4. Equipment………………………………………………………………..119

3.4.5. Modeling…………………………………………………………………120

3.4.6. Synthesis of DNA-Functionalized Silica Particles………………………123

3.4.7. Determining Number of Oligos Per Silica Particle………………………124

3.4.8. Atto647N Conjugation to Target Strands………………………………..125

3.4.9. Flow Cytometry Assay and Analysis to Measure Target Binding………125

3.4.10. Fluorescence Microscopy of Atto647N-Labeled Targets Hybridized to Beads……………………………………………………………………………126

3.5. Appendix…………………………………………………………………………...127

3.6. References………………………………………………………………………….152

Chapter 4. Summary and Future Outlook…………………………………………………..155

4.1. Summary…………………………………………………………………………...156

4.2. Future Outlook for Multivalent DNA Hybridization………………………………158

4.2.1. Further Applications for Multivalent DNA Hybridization in Diagnostics……………………………………………………………………...158

4.2.2. Investigating HeteroMV DNA Hybridization on a Lipid Surface……….163

4.2.3. Potential Advantages and Obstacles for Use of HeteroMV DNA Hybridization in Therapeutic Applications……………………………………..165

4.3. References………………………………………………………………………….166

 

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, General Chemistry, Physical Chemistry, Biochemistry
Mot-clé	Nucleic Acid Hybridization Multivalent Binding
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	James Kindt, Emory University Jennifer Heemstra, Emory University Yonggang Ke, Emory University

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