DNA Origami in Protein Oligomer Assembly & Mesoscale Self-assembly of DNA Origami Nanorod with Polynucleotide Brush Restricted; Files Only

Abstract

Over the last two decades, research in DNA nanotechnology has seen astonishing growth and has yielded exquisite DNA-based nanostructures that span a broad range of sizes and complexity. DNA origami nanostructures (DONs) have been widely investigated for numerous biomedical applications. This dissertation describes an application using DNA origami to guide protein complexes assembly, and construction of larger DNA nanostructure. Chapter 1 focus on developing a method for controlling the assembly of protein oligomers on DNA nanostructures using a strand replacement strategy, resulting in the production of different protein oligomers. Chapter 2 and 3 both describe the combination of surface-initiated enzymatic polymerization with DNA origami to create stable, polynucleotide brush-functionalized structures can be precisely controlled in terms of composition, morphology, and site-specific location of initiation sites on the origami core. The resulting structures have potential applications in drug delivery and other nanoscale delivery systems. Later, Chapter 4 and 5 include some other work during my PhD journey.

Table of Contents

Chapter 1. Controlling Protein Assembly by Sequentially Regulating Proximity and Valency with A DNA Origami Template 1 

1.1Abstract 2

1.2 Introduction 2

1.3 Results and Discussion 5

  1.3.1 (ald-DNA)3 Preparation 5

     1.3.2 DNA origami formation & Attachment of ald-DNA to the origami 6

     1.3.3 ssDNA (on ald) and Connector Length Modification 8

     1.3.4 Connect & Release Ald Dimer 10

     1.3.5 Attachment Manner Modifications for Trimer and Tetramer Assembly 12

     1.3.6 Connect & Release Linear Trimer and Triangle 15

     1.3.7 Connect & Release Linear Tetramer, Square and Shape Y 17

1.4 Conclusion 20

1.5 Materials and Methods 22

1.5.1      Materials and supplies 22

1.5.2      Protein expression, modification and characterization 27

1.5.3      4HB origami and sticky ends for dimerization design 29

1.5.4      Random protein connection & Blocker deactivation of the extra connector 30

1.5.5      Connector ratio test, protein distances test, and protein multimer gel result 32

1.5.6      Protein multimer yield calculation 34

1.5.7      Distance between alds calculation 41

1.5.8      Pathway of the linear tetramer formed in only one connection step 43

1.6 References 44

Chapter 2. Programmable Site-Specific Functionalization of DNA Origami with Polynucleotide Brushes 46

  2.1 Abstract 47

  2.2 Introduction 47

  2.3 Results and Discussion 49

  2.4 Conclusion 60

  2.5 Acknowledgements 61

  2.6 Keywords 61

  2.7 Materials and Methods 62

  2.7.1 Materials 62

2.7.2 DNA Origami Preparation, Modification and Characterization 62

     2.7.3 Computational Section 67

     2.7.4 Supporting Figures 69

     2.7.5 Supporting Table 88

  2.8 References 88

 

Chapter 3. Spatiotemporal Control of Polynucleotide Brush Growth and Mesoscale Self-assembly of DNA Origami Nanorod 92

  3.1 Abstract 93

  3.2 Introduction 94

  3.3 Results and Discussion 95

3.3.1      Spatiotemporal Control of Polynucleotide Brush Growth on DNA Origami 95

3.3.2      Bi-functional Modification Through Sequential Polymerization 99

3.3.3      Spatio Control of DNA Origami Mesoscale Self-assembly 101

  3.4 Conclusions 105

  3.5 References 105

 

Chapter 4. Seeded DNA Origami Assembly Pathway Study 108

  4.1 Abstract 109

  4.2 Results and Discussion 109

     4.2.1 4HB ends & 1/3 seeds comparison 109

     4.2.2 6HB assembly study 113

     4.2.3 FRET (fluorescence resonance energy transfer) experiments 118

  4.3 Methods and Technologies 119

 

Chapter 5. Other Contributions 121

 

 

 

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Stichwort	DNA origami DNA nanotechnology
Committee Chair / Thesis Advisor	Yonggang Ke, Emory University
Committee Members	David Lynn, Emory University Vincent Conticello, Emory University

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