Deoxyribozyme Nanoparticle Nanozymes: from Splicing to Mechanism Open Access

Abstract

Nanozymes – created by attaching enzymes to a nanoscale scaffold – are just now coming into their full potential as tools for RNA knockdown and splicing. In Chapters 1-2 of this thesis, we review both gene knockdown technologies and gold nanoparticle-based nanozymes, with emphasis on deoxyribozyme (DNAzyme) nanoparticle (DzNPs) constructs. DzNPs are a subclass of nanozymes that have potential applications ranging from RNA gene regulation to analyte sensing. In Chapter 3, we focus on discovering the mechanism of action of DzNPs inside cells. To address this goal, we report the construction of core-attached DzNPs that are inactive until the DNAzymes are released from their scaffold, leading to a 51-fold increase in DNAzyme activity. Our preliminary data suggests there is gene knockdown by core-attached DzNPs in cells. Since this activity must come from the released DNAzymes, this experiment gives us a window into the mechanism of action of these constructs. In Chapter 4, we characterize DzNPs targeting GATA-3 mRNA, measuring their activity, longevity and stability. We found that these DzNPs can last for up to 9 months when stored in excess salt and DNAzyme; and they are most active when synthesized via the freezing method, rather than the salt aging method. Subsequently in Chapter 5, we discuss the building of a new type of nanozyme comprised of DNAzyme and RtcB ligase subunits attached to a gold nanoparticle core that can splice virtually any RNA stem-loop. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. We conclude our studies in Chapter 6 with work exploring methods to show RNA splicing inside cells, as a future step. These splicing nanozymes, as well as DzNPs for gene knockdown, represent a new promising approach for gene manipulation that has potential for in vivo applications.

Table of Contents

Table of Contents

Chapter 1: Introduction on Gene Knockdown

1.1. Introduction ................................................................................................................. 1

1.2. Importance of Gene Knockdown .................................................................................... 1

1.3. Methods of Gene Knockdown ........................................................................................ 2

1.3.1. Antisense Oligonucleotides ................................................................................... 2

1.3.2. Ribozymes ........................................................................................................... 4

1.3.3. RNA Interference .................................................................................................. 9

1.3.4. CRISPR .............................................................................................................. 11

1.4. Gene Knockdown with DNAzymes ............................................................................... 15

1.4.1. Why Use DNAzymes ......................................................................................... 15

1.4.2. History of RNA-cleaving DNAzymes ................................................................... 16

1.4.3. Second Generation of DNAzymes: Birth of 10-23 and 8-17 ................................ 19

1.4.4. Applications of RNA-cleaving DNAzymes ........................................................... 22

1.4.5. Motivation: Our System, the DzNP Nanozyme ................................................... 23

1.5. References ................................................................................................................... 25

Chapter 2: Introduction of Nanozymes

2.1. Competing Definitions of “Nanozyme” .......................................................................... 35

2.2. Advantages of Using a Gold Core ................................................................................ 40

2.2.1. Ease of Synthesis ............................................................................................... 40

2.2.2. Biocompatibility and Low Toxicity Profile ............................................................ 41

2.2.3. Use of Gold as a Reporter: Labeling, Sensing, Staining ..................................... 42

2.2.4. Functionalization and Cellular Uptake ................................................................ 42

2.2.5. Use of AuNPs in this Study ................................................................................. 43

2.3. Examples of Gold Core Nanozymes ............................................................................. 44

2.3.1. Nanozymes Functionalized with Chemical Ligands ............................................ 44

2.3.2. Spherical Nucleic Acids ...................................................................................... 44

2.3.3. Nanozymes Functionalized with Nucleic Acids and an Enzyme .......................... 48

2.3.4. 10-23 DNAzyme Nanoparticle ............................................................................ 49

2.3.5. Uranyl-specific 39E DNAzyme Nanoparticle ....................................................... 51

2.3.6. Nanoscript: Transcription Factor Nanoparticle Mimic .......................................... 51

2.3.7. Ribozyme Nanoparticle ...................................................................................... 53

2.3.8. CRISPR-gold Nanozyme .................................................................................... 54

2.3.9. Transition Metal Catalyst Functionalized Nanozymes ......................................... 55

2.4. References ................................................................................................................... 57

Chapter 3: Mechanism of DNAzyme Nanoparticles (DzNPs) for RNA Knockdown

3.1. Introduction .................................................................................................................. 69

3.2. Optimizing DzNP Synthesis .......................................................................................... 77

3.3. Synthesizing Inactivated DNAzyme Nanoparticles ....................................................... 78

3.4. Testing Whether DzNPs Degrade mRNA While Intact .................................................. 80

3.5. Proposed Experiments for Future Work ........................................................................ 81

3.6. Conclusion ................................................................................................................... 83

3.7. Materials and Methods ................................................................................................. 84

3.8. Appendix ...................................................................................................................... 91

3.9. References ................................................................................................................... 93

Chapter 4: Gene Knockdown of GATA-3

4.1. Introduction .................................................................................................................. 98

4.2. In Vitro Activity of GATA-3 Targeted DzNPs ............................................................... 101

4.3. Characterization of GATA-3 2251 DzNPs ................................................................... 105

4.4. Conclusion ................................................................................................................. 109

4.5. Materials and Methods ............................................................................................... 110

4.6. References ................................................................................................................. 114

Chapter 5: Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle

5.1. Introduction ................................................................................................................ 119

5.2. Results and Discussion .............................................................................................. 122

5.2.1. RtcB Is More Active on Stem-Loops Than Linear RNA ..................................... 122

5.2.2. DNAzymes and RtcB Splice an RNA Stem-Loop .............................................. 125

5.2.3. Dz1Dz2NP Splicing Using Excess RtcB ............................................................. 127

5.2.4. Nanozyme Synthesis ........................................................................................ 127

5.2.5. Nanozyme Conjugates Splice RNA Stem-Loop Targets.................................... 129

5.3. Conclusion ................................................................................................................. 132

5.4. Materials and Methods ............................................................................................... 134

5.5. Author Contributions and Acknowledgements ............................................................ 143

5.6. Supplementary Materials and Methods ...................................................................... 144

5.7. Supplementary Figures .............................................................................................. 150

5.8. References ................................................................................................................. 167

Chapter 6: Cellular Splicing – Using DzNPs to Splice the XBP1 intron

6.1. Introduction ................................................................................................................ 171

6.2. Results and Discussion .............................................................................................. 174

6.2.1. Modifying the Construct ................................................................................... 174

6.2.2. Control Experiment: Transfecting Original Plasmid .......................................... 175

6.2.3. Comparing Modified Plasmid to Original Plasmid ............................................. 177

6.3. Future Work ................................................................................................................ 180

6.4. Other Systems to Detect Splicing in Cells ................................................................... 180

6.5. Conclusion .................................................................................................................. 181

6.6. Materials and Methods ............................................................................................... 182

6.7. References ................................................................................................................. 187

Chapter 7: Perspectives and Future Work

7.1. Mechanism of DzNPs ................................................................................................. 191

7.2. In Vitro RNA Splicing by DzNP-Nanozymes ............................................................... 192

7.3. Cellular RNA Splicing by DzNPs ................................................................................ 193

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Nanotechnology Engineering, Biomedical
Keyword	deoxyribozyme nanozyme DNAzymes RtcB gold nanoparticle synthetic biology
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	Stefan Lutz, Emory University David Lynn, Emory University

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