Fundamental properties and applications of surface confined enzymes in gene regulation and molecular motors Open Access

Yehl, Kevin Matthew (2015)

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The vast majority of enzymes function in confined environments within or near lipid membranes, or within a supramolecular assembly containing many biomolecules. Examples range from receptor tyrosine kinases to motor proteins and the ubiquitous enzymes involved in DNA replication and protein synthesis. Confined enzymatic reactions are also widely employed in clinical chemistry and biotechnology. Despite its prevalence, we still have a limited ability to predict and control the properties of catalytic reactions upon confinement of the enzyme and substrate. The major goal of this thesis is to study the properties of confined catalytic reactions and to take advantage of insights for improved therapeutic and bio-sensing applications.

In Chapter 1, we describe the historical context and summarize representative literature on the topic of surface confined catalytic reactions. The chapter includes a discussion of general properties of catalytic reactions in comparison to solution, and the theoretical underpinning of differential properties on surfaces.

Chapter 2 describes our efforts to develop and test model systems to study the impact of catalyst immobilization. DNAzymes, or catalytic oligonucleotides, were immobilized onto gold nanoparticles and used as a model system to study the effects of enzyme packing density and orientation, linker length and composition, and passivation on activity. From these findings, design rules were developed to synthesize highly active DNAzyme nanoparticles, which were shown to readily enter mammalian cells, possess enhanced nuclease resistance and catalytic activity, and can regulate gene expression in a dose dependent manner. Importantly, in this chapter, we showed that DNAzyme nanoparticle conjugates down-regulate GDF15 mRNA expression by 60% in a cell culture model. GDF15 has clinical relevance for the treatment of many types of cancers, and particularly Herceptin® resistant metastatic breast cancer.

In Chapter 3, we expanded upon these studies in a more therapeutically relevant model to show that DNAzyme nanoparticles are highly modular and can regulate TNFα protein expression by targeting the corresponding mRNA in a rat model. The goal of these studies was to develop an anti-inflammatory therapy to prevent heart failure post cardiac infarction. The hypothesis was that the resulting tissue damage caused by cardiac infarction is due to an over-active immune response (up-regulation of TNFα), which could be minimized by down-regulating TNFα. We showed that DNAzyme nanoparticles targeting TNFα were more efficient when compared to siRNA or to antisense nanoparticles. In vivo experiments using a rat model of cardiac infarction showed that animals treated with DNAzyme nanoparticles targeting TNFα mRNA had a ~40% improvement in heart function, as determined by fractional shortening measurements of the rat's infarct tissue.

Chapter 4 describes the study of surface-immobilized catalytic reactions as models for molecular machines and motors. Following earlier results demonstrating the therapeutic potential of DNAzyme nanoparticles, we wondered how reactions would proceed if the substrate was surface immobilized. We anticipated that the particles would move along the surface to maximize Watson-Crick base pairing in a ‘burnt bridge' mechanism of translocation. We chose to investigate the RNase H enzyme, which catalyzes the hydrolysis of RNA in RNA-DNA duplexes at orders of magnitude greater efficiency than DNAzymes. In these studies, we showed that multivalent DNA modified particles (diameter = 5 µm, 107 DNA/particle) move along an RNA-monolayer surface in the presence of RNase H and translocate through a unique rolling mechanism. Thus, we termed these motors DNA monowheels (DMWs). This unique mechanism of translocation produces emergent properties enabling the motors to reach rapid speeds up to 5 µm/min and processivity of several hundreds of µm's, thus surpassing the fastest synthetic DNA-based motor by three orders of magnitude and approaching the capabilities of biological motor proteins. Interestingly, these motors displayed the first experimental example of self-avoiding diffusion. Due to the rolling mechanism of motion, we were able to program DMWs to move in a linear fashion by incorporating anisotropy into the particle "chassis" structure. Accordingly, this was the first example of directional motion without the influence from an external field or patterned track. Because of the motor robustness, size, and molecular sensitivity, we showed that DNA monowheels could be used to detect single nucleotide mutations using a simple smartphone readout, thus demonstrating the simplicity and usefulness of DNA-based motors towards real world applications.

Finally, Chapter 5 provides a perspective discussing the potential for studying confined multi-enzyme model systems to more accurately recapitulate living systems. Such constructs are anticipated to have emergent, synergistic properties that impact the fields of sensing and gene editing technologies.

Table of Contents

Chapter 1: Properties and applications of surface confined enzymes. 1

1.1 Introduction. 2

1.1.1 Prevalence throughout biology. 2

1.2 Theory on surface confinement of enzymatic reactions. 2

1.2.1 Immobilization techniques. 3

1.2.2 Stability. 4

1.2.3 Enhanced catalysis. 5

1.3 Prevalence of surface confined enzymatic reactions. 6

1.3.1 Industrial. 6

1.3.2 Bio-medical Applications. 7

1.3.3 Synthetic Motors. 9 Seminal studies. 10 Role of surface confined enzymatic reactions on the progress of molecular motors. 12 Burnt bridge mechanism. 12 DNAzyme based motors. 12 Endonuclease powered motors. 18

1.4 Aim and Scope. 22

1.5 References. 23

Chapter 2: Nanoparticle Immobilized Deoxyribozymes: Model system for studying enzyme confinement and RNAi-independent gene regulation. 29

2.1 Introduction. 30

2.2 Results and Discussion. 33

2.2.1 DzNP synthesis and catalysis. 33

2.2.2 Role of surface density and linker chemistry on catalysis. 35

2.2.3 Role of DNAzyme orientation on catalytic activity for DzNP. 38

2.2.4 Photo-thermal activation of DNAzymes. 40

2.2.5 DzNP cellular uptake and resulting gene regulation. 41

2.3 Conclusions. 46

2.4 Materials and Methods. 46

2.4.1 DNA sequences. 46

2.4.2 Synthesis of gold nanoparticles 47

2.4.3 Preparation of DzNPs. 47

2.4.4 TEM imaging of DzNPs. 48

2.4.5 Calculation of the number of deoxyribozyme molecules per AuNP. 48

2.4.6 Measurement of DzNP activity. 50

2.4.7 Determination of nuclease resistance. 51

2.4.8 Cell culture and DzNP mediated gene knockdown. 51

2.4.9 Chemical activation of DNAzyme catalytic activity (thiol displacement). 52

2.4.10 Photo-thermal activation of DNAzymes. 52

2.4.11 Mercaptoethanol (ME) passivation of DzNPs. 54

2.5 References. 54

Chapter 3: In vivo efficacy of DNAzyme nanoparticles as an anti-inflammatory therapy following myocardial infarction. 62

3.1 Introduction. 63

3.2 Results and Discussion. 67

3.2.1 Synthesis and characterization of DzNPs. 67

3.2.2 Internalization of DzNPs by macrophages and myocytes in vitro. 68

3.2.3 DzNPs in vitro cytotoxicity. 70

3.2.4 In vitro TNFα knockdown. 71

3.2.5 Ex vivo biodistribution of DzNPs determined by fluorescence imaging. 73

3.2.6 In vivo activity of DzNPs towards down regulation of TNFα 74

3.3 Conclusion. 79

3.4 Material and Methods. 80

3.4.1 Oligonucleotide sequences. 80

3.4.2 Gold nanoparticle synthesis. 81

3.4.3 DzNP synthesis. 81

3.4.4 Quantification of DNAzyme loading density on gold nanoparticles. 83

3.4.5 Cell culture. 84

3.4.6 DzNP uptake in macrophages and myocytes. 84

3.4.7 In vitro silencing of TNFα with DzNPs. 85

3.4.8 DzNP in vitro cytotoxicity. 85

3.4.9 Myocardial infarction and particle injection. 86

3.4.10 In vivo imaging. 86

3.4.11 Echocardiography and invasive pressure-volume hemodynamics analysis. 87

3.4.12 In vivo gene expression and plasma cytokine analysis. 87

3.4.13 Statistics. 88

3.5 References. 88

Chapter 4: Rolling DNA-based motors with superdiffusive transport powered by RNase H. 97

4.1 Introduction. 98

4.2 Results and Discussion. 100

4.2.1 Synthesis and stability of the RNA monolayer. 100

4.2.2 Kinetic analysis of RNase H hydrolysis of surface immobilized RNA and the role of passivation. 103

4.2.3 Synthesis and characterization of DNA functionalized 5 µm diameter silica Particles. 105

4.2.4 Characterization of particle translocation. 107

4.2.5 Experimental realization and computation models of superdiffusive transport. 108

4.2.6 Mechanism of particle translocation. 110

4.2.7 Linear transport of RNase H powered DNA monowheels. 121

4.2.8 SNP detection through measuring DNA monowheel displacements. 125

4.3 Conclusions. 127

4.4 Materials and Methods. 128

4.4.1 Theory and simulation of self-avoiding particle rollers. 128 True self-avoiding walk. 128 Multivalency and enzyme kinetics. 129 Displacement distribution. 132 Velocity estimate. 133

4.4.2 Displacement distributions from particle tracking. 134

4.4.3 Power conversion efficiency. 135

4.4.4 Materials. 136

4.4.5 Oligonucleotide sequences. 137

4.4.6 Optical Microscopy. 138

4.4.7 Super resolution imaging of the fluorescence depletion tracks. 138

4.4.8 Thermal evaporation of gold films. 139

4.4.9 Fabrication of RNA monolayers. 140

4.4.10 Determining RNA surface density. 140

4.4.11 µ-contact printing of RNA tracks. 141

4.4.12 Synthesis of azide functionalized particles. 141

4.4.13 Synthesis of high DNA density silica particles. 142

4.4.14 Determining DNA particle surface density. 142

4.4.15 Determination of RNase H surface kinetics. 143

4.4.16 RNase H powered particle translocation. 143

4.4.17 Smartphone based SNP detection. 145

4.5 References. 146

Chapter 5: Conclusion and Perspectives. 151

5.1 Summary. 152

5.1.1 DzNP model systems and their applications towards gene regulation. 152

5.1.2 DNA based monowheels. 153

5.2 Future Outlook. 154

5.2.1 Other Enzymatic reactions. 154

5.2.2 Multi-enzyme reactions. 155 Applications towards sensing. 155 Applications towards gene editing. 156

5.3 References. 157

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