Harnessing DNA Motors for Sensing and Information Processing Applications Restricted; Files Only

Abstract

Molecular motors exist ubiquitously in biological systems, performing diverse roles by converting chemical energy into mechanical work. The ability of these complex molecular machines to catalyze energy transduction at the nano-scale has inspired scientists to develop synthetic analogues capable of mimicking these vital properties. This thesis aims to explore the potential of utilizing DNA nanotechnology to design synthetic molecular machines, termed here as DNA motors, that can be programmed to serve as a new platform for far-from-equilibrium chemical sensing.

The first chapter provides historical context and discusses leading advancements in structural and dynamic DNA nanotechnology, including the classification and applications of various DNA machines and nanodevices. The second chapter presents our work on the development of programming DNA motors to sense, process, and respond to chemical input. Here we introduce DNA motors capable of carrying out logic gate operations in response to specific chemical input signals.

The third and fourth chapters delve further into the sensing applications of these DNA motoes as we focus on their use to detect viral proteins and develop methods by which these motors can produce easily detectable output signals. With this approach, we present a novel method of detecting and responding to different viruses utilizing our DNA motors.

In chapter five, we discuss our design of DNA motors capable of responding to UV light, which further expands the versatility of these nanodevices from strictly chemical cues to also physical stimuli. We share how the UV-responsive DNA motors provide additional opportunities for controlled activation and operation. Finally, we conclude in chapter six with a comprehensive summary of our work, highlighting its potential impact on the fields of synthetic biology, nanotechnology, and chemical sensing. We also share possible research directions and applications for these programmable DNA nanomachines in the future.

This dissertation provides an extensive exploration of how DNA nanotechnology can be harnessed to develop advanced molecular motors for various sensing applications. Overall, the work contributes valuable knowledge and tools to the ongoing endeavor of designing synthetic molecular machines with the sophisticated functionalities found in biological systems.

 

Table of Contents

Table of Contents

Chapter 1. Properties and applications of DNA-based nanomachines

1.1 Biological motor proteins: unraveling nature's nanomachinery

1.2 DNA-based machines

1.2.2 DNA-based switches

1.2.3 DNA-based machines

1.2.3.1 DNA walkers

1.2.3.2 Polyvalent DNA-based motors

1.4 Applications

1.5 Aim and scope

1.6. References

Chapter 2. Chemical-to-mechanical molecular computation using DNA-based motors with onboard logic

2.1. Introduction

2.2. Results and Discussion

2.2.1. Design of DNA-based motors with onboard logic (DMOLs)

2.2.2. Computation of AND gate

2.2.3. Computation of OR gate

2.2.4. Multiplexing fluorophore-encoded DMOLs

2.2.5. DMOL-to-DMOL networking through cascading logic gates

2.2.6. Multiplexing with DMOLs by size and material

2.3. Conclusions

2.4. Materials and methods

2.4.1. Materials

2.4.2. Microscopy

2.4.3. Thermal evaporation of gold films

2.4.4. Fabrication of RNA monolayers

2.4.5. Synthesis of azide-functionalized DMOLs

2.4.6. Synthesis of high-density DNA silica and polystyrene DMOLs

2.4.7. Particle translocation

2.4.8. Image processing and particle tracking

2.7. Appendix

2.8. References

Chapter 3. Rolosense: Mechanical detection of SARS-CoV-2 using a DNA-based motor

3.1. Introduction

3.2. Results and Discussion

3.2.1. Design principles of Rolosense platform

3.2.2. Detecting SARS-CoV-2 in artificial saliva

3.2.3. Multiplexed detection of SARS-CoV-2 and Influenza A viruses

3.2.4. Detecting SARS-CoV-2 via smartphone microscope readout

3.2.5. Detecting SARS-CoV-2 in breath condensate generated samples

3.3. Conclusions

3.4. Materials and methods

3.4.1 Materials

3.4.2. Microscopy

3.4.3. Viruses

3.4.4. Cells and plasmids

3.4.5. Virus-like particles (VLPs) production and characterization

3.4.6. Quantification and imaging of VLPs using single particle microscopy imaging

3.4.7. Thermal evaporation of gold films

3.4.8. Fabrication of RNA/DNA aptamer monolayers

3.4.9. Synthesis of azide-functionalized motors

3.4.10. Synthesis of high-density DNA silica and polystyrene motors

3.4.11. Preparation of antibody coated motors and chips

3.4.12. Breath condensate collection

3.4.13. Motor translocation

3.4.14. Preparation and testing of blinded LoD challenge panel

3.4.15. Image processing and particle tracking

3.5. Appendix

3.6. References

Chapter 4. Fuel-free Rolosense: using DNA-coated Brownian particles for rapid multiplexed detection of respiratory viruses

4.1. Introduction

4.2. Results and Discussion

4.2.1. Developing FF-Rolosense assay

4.2.2. Optimizing sensitivity of FF-Rolosense assay by changing aptamer span length

4.2.3. Multiplexed detection using influenza A virus

4.2.4 Using 3D-printed brightfield imager to readout FF-Rolosense performance

4.3. Conclusions

4.4. Materials and Methods

4.4.1. Materials

4.4.2. Microscopy

4.4.3. Viruses

4.4.5. Thermal evaporation of gold films

4.4.6. Fabrication of DNA aptamer monolayers

4.4.7. Synthesis of azide-functionalized particles

4.4.8. Synthesis of high-density DNA silica and polystyrene particles

4.4.9. Breath condensate collection

4.4.10. Particle translocation

4.4.11. Image processing and particle tracking

4.5. Appendix

4.6. References

Chapter 5. On-demand photoactivation of DNA-based motor motion

5.1. Introduction

5.2. Results and discussion

5.2.1. Design of UV-activated DNA motors

5.2.2. Light-controlled motion of DNA-based motors

5.2.3. DNA-based motors as programmable, light-responsive bio-mechanical machines

5.3. Conclusions

5.4 Materials and Methods

5.4.1 Materials

5.4.1.1. Oligonucleotides

5.4.1.2. Reagents

5.4.1.2. Equipment

5.4.2. Methods

5.4.2.1. Thermal evaporation of gold films

5.4.2.2. Fabrication of RNA monolayers

5.4.2.3. Synthesis of azide-functionalized particles

5.4.2.4. Synthesis of high-density DNA silica motors

5.4.2.5. Particle translocation

5.4.2.6. Image processing and particle tracking

5.5. Appendix

5.6. References

Chapter 6. Summary and outlook

6.1. Summary

6.2. Outlook and perspective

6.2.1. Enhancing the chemical-to-mechanical transduction efficiency of DNA motors

6.2.2. Further developments to optimize FF-Rolosense assay

6.3. Concluding remarks

6.4. References

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, General Nanotechnology Chemistry, Analytical
Palavra-chave	Chemical sensing DNA computation Biosensors DNA machines DNA motors
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	Yonggang Ke, Emory University Vincent Conticello, Emory University

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