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DEVELOPMENT OF GENETICALLY ENCODABLE FLUOROGENIC RNA SENSORS FOR SMALL MOLECULE DETECTION IN LIVE CELLS

Abstract
Fluorescence imaging of intracellular small molecules in live cells is crucial to understanding various biological pathways and disease-related phenomena. It enables researchers to learn more in-depth about how certain metabolisms and signal transducing pathways function, and it could provide valuable insights to the therapeutic and biomedical field. Fluorogenic RNA-based sensors have been recently developed and widely used in detecting small molecules in live cells. They exhibit general advantages over other traditional fluorescence imaging-based detection methods, such as being genetically encodable, highly modular, readily programmable, and able to develop specificity towards potentially any target of interest. These merits make fluorogenic RNA-based sensors a superior tool to discover the unexplored area in cell biology. In this thesis, we will show several examples of how these fluorogenic RNA sensors are developed and applied in live cells to study and reveal previously unknown information on the role of the target small molecules in the cellular systems. First, in order to better understand the role of the alarmone, guanosine tetra-and pentaphosphate (p)ppGpp, in the stringent response in bacteria, we adapted the ppGpp recognition module from its naturally occurring riboswitch and engineered it into a fluorescent sensor by fusing it with a fluorogenic RNA aptamer, Broccoli. As a result, we were able to apply our sensor in E. coli and monitor the cellular dynamics of (p)ppGpp in live cells for the first time in over half a decade since its discovery. Second, it is hypothesized that (p)ppGpp and another bacterial second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) synergistically support certain physiological processes in bacteria. However, no direct correlation between the two has been proved or disproved. To further study the interactions between the two signaling molecules, we developed a multiplexed sensor to detect (p)ppGpp and c-di-GMP simultaneously. Third, to image beyond two or three targets in the same cells, one must overcome the challenge of lacking spectrally resolvable fluorophores that can be imaged simultaneously. Without further expanding the current imaging spectrum, we managed to circumvent the issue by exploring the possibility of sequential imaging. Here, we constructed the “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES) and adopted an “imaging-washing cycle” method to achieve multiplexed and orthogonal detection of small molecules, S-Adenosyl-L-methionine (SAM) and S-Adenosyl-L-homocysteine (SAH). Finally, to demonstrate the universal applicability of the fluorogenic RNA-based sensors in both prokaryotes and eukaryotes, we developed a (2’-5’, 3’-5’) cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP) sensor for its detection in live mammalian cells.
Type
campusfive
article
dissertation
Date
2023-09-01
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http://creativecommons.org/licenses/by-nc-nd/4.0/
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