Engineering synthetic ligand-responsive RNA devices for controlling the cell cycle
Abstract/Contents
- Abstract
- Synthetic biology aims to program cells by applying engineering principles to the design of genetic circuits to control and monitor cellular behavior. Programmed cells can harness existing dynamically coordinated biologically processes to rescue cellular malfunctions, control existing cellular morphology, and introduce useful novel functionality. Recent advances in the field are leading to genetic circuits of higher-order complexity in mammalian cells, which may ultimately be used to study and reprogram phenotypes that play a key role in human health and disease, such as those linked to the cell cycle. RNA-based control devices can provide specific and modular control of gene expression in response to endogenous or exogenous inputs in living cells. Specifically, we show that for a ribozyme-based device platform, ribozyme switches previously prototyped in yeast are able to regulate gene expression in a predictable (R-squared = 0.63--0.97), ligand-responsive manner in human HEK 293, HeLa, and U2-OS cell lines without any change to device sequence nor further optimization. We also show that these simple, general-purpose components (an RNA ribozyme coupled to an RNA aptamer) are a compact (~200 nt) and effective way to control a complex phenomenon such as arresting up to 80% of a population of mammalian cells in the G2/M phase of the cell cycle. Current methods for altering cell cycle progression can present a number of limitations, including exhibiting specificity towards particular cell types, being broadly disruptive of cellular processes, and being limited in capacity to extend to different networks. A genetically encoded system supporting control over cell cycle progression has the potential to address limitations with existing chemical approaches. Further, these switches are modular and readily adapted to arrest cells in the G0/1 phase of the cell cycle and in response to at least two different small molecule inputs. This work describes the first example of the application of synthetic RNA-based gene-control devices to the regulation of cell cycle progression in human cells. We further show that siRNAs can induce the arrest of > 90% of a population of cells in the G0/1 phase of the cell cycle. This indicates the potential to build miRNA-based devices to control the cell cycle that complement the ribozyme-based approaches. Thus, ligand-responsive RNAs represent a class of synthetic biology tools that are both genetically encoded and capable of regulating intracellular protein levels in response to user-specified molecular signals, and can be adapted for sophisticated control of complex processes in human cells.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Wei, Kathy Yufeng |
|---|---|
| Associated with | Stanford University, Department of Bioengineering. |
| Primary advisor | Smolke, Christina D |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Skotheim, Jan, 1977- |
| Thesis advisor | Swartz, James R |
| Advisor | Skotheim, Jan, 1977- |
| Advisor | Swartz, James R |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Kathy Yufeng Wei. |
|---|---|
| Note | Submitted to the Department of Bioengineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Kathy Y Wei
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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