Developing Nucleic Acid-Based Biosensor Platforms
Open Access
- Author:
- Vezeau, Grace
- Graduate Program:
- Agricultural and Biological Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 24, 2021
- Committee Members:
- Jeffrey Catchmark, Major Field Member
Howard Salis, Chair & Dissertation Advisor
Philip Bevilacqua, Outside Unit Member
Paul Heinemann, Program Head/Chair
Andrew Zydney, Outside Field Member - Keywords:
- synthetic biology
biosensors
RNA
riboswitch
CRISPR-Cas9
cell-free expression systems
biophysics
nucleic acid secondary structure - Abstract:
- Biological systems are excellent sensing platforms, able to specifically detect a diverse array of chemical and biomolecular stimuli in their environment and respond rapidly and appropriately. Cells accomplish this by using genetically-encoded sensing systems, where a gene product such as protein or nucleic acid binds to a specific compound and subsequently directs a change in the expression of a relevant gene. While these genetically-encoded sensing systems let cells respond to their environments and maintain homeostasis, these responses are rarely human-usable. The process of re-engineering biological sensors is further complicated by the need for recognition elements for novel inputs and the fact that biological recognition elements are rarely modular. While re-engineering protein-based, genetically-encoded sensing elements is simple in a few cases, it is largely a complex and time-consuming process. Developing new nucleic acid-based recognition elements and mechanisms of signal transduction, however, is enabled by their relatively smaller sequence space and more predictable sequence-structure relationship. Here, I develop new classes of nucleic-acid based sensors via computational sequence design, expanding the capabilities of nucleic acid-based sensing and developing sensors with the potential for real-world utility. I first focus on developing protein-detecting RNA switches that operate in cell-free expression systems. To enable further development of the cell-free platform, I examine how cell-free reaction formulation affects the processes of gene expression, and overall output characteristics. I next examine sequence-function relationships of mutated single-guide RNAs (sgRNAs) in directing Cas9 activity. Finally, I use the learned design rules to develop Cas9-based sensors that directly detect viral RNA sequences.