Building Nucleic Acid-Based Technologies for Biological Sensing and Modulation Open Access

Manuel, Brea (Fall 2022)

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Nucleic acids are historically known and studied for their natural functions of information storage. However, over the last few decades, functional nucleic acids have emerged in the fields of biotechnology, bioremediation, and biomedicine, as they possess the abilities to bind targets of interest and perform catalysis. Aptamers have been evolved to bind several targets, including proteins, heavy metals, and small molecules. As a result, many different aptamer-based applications have come to fruition. This dissertation aims to explore various applications of aptamers and develop different technologies for biological sensing and modulation, advancing the fields of biotechnology and biomedicine. Chapter 1 introduces the different functional nucleic acids, their uses, and the gaps in the field. In Chapter 2, we sought to address current challenges of modulating aptamers to produce stable nucleic acids that are capable of binding a range of targets with a range of affinities. We then use these aptamers in Chapter 3 in an attempt to study sequestration of small molecules, and the effects of this phenomenon on enzymes. Specifically, we use these aptamers to enhance enzyme activity with total control of the various kinetic parameters. In Chapter 4, we take an opposite turn and explore reversible control of enzymes and functional nucleic acids using a thermoreversible protecting/caging group. In Chapter 5, we then use the idea of aptamer evolution to explain the effects of the selection process on diversity, equity, and inclusion. Lastly, we summarize our findings and propose future explorations of aptamers and their applications in Chapter 6.

Table of Contents

Chapter 1: Introduction. 1

1.1 Functional Nucleic Acids 1

1.2 DNAzymes 2

1.3 Aptamers 3

1.4 Modulating Aptamer Recognition with Base Modifications 4

1.6 Aptamer-Based Small Molecule Sequestration. 6

1.7 Summary and Conclusions of this Dissertation. 8

Chapter 2: Systematically Modulating Aptamer Affinity and Specificity by Guanosine-to-Inosine Substitution21 10

2.1 Abstract 10

2.2 Introduction. 11

2.3 Results and Discussion. 13

2.4 Conclusion. 20

2.5 Materials and Methods. 22

Chapter 3: Modular Catalysis: Aptamer Enhancement of Enzyme Kinetics in a Nanoparticle Reactor 24

3.1 Abstract 24

3.2 Introduction. 24

3.3 Results and Discussion. 27

3.4 Conclusions. 35

3.5 Materials and Methods. 36

Chapter 4: Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging. 41

4.1 Abstract 41

4.2 Introduction. 42

4.3 Results and Discussion. 43

4.4 Conclusions. 62

4.5 Materials and Methods. 63

Chapter 5: Recruit and Retain a Diverse Workforce159 85

5.1 Abstract 85

5.2 Introduction. 85

5.3 Assessing Candidates. 86

5.4 Origins of Exclusivity. 89

5.5 Cultivating Inclusion. 91

5.6 Outlook. 92

Chapter 6: Conclusions and Future Perspectives 93

Appendix A: Omitted Data from Chapter 221 98

Appendix B: Omitted Data from Chapter 3. 109

Appendix C: Omitted Data from Chapter 4. 119

References. 155


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