Development and Optimization of Novel Tools for Exploring RNA-Based Cell-Cell Communication
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Duong, Cindy T
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Abstract
Cells communicate by sending and receiving messages that are necessary to carry out essential functions. In cancer, this intricate signaling network of interactions may help tumor cells develop resistance to treatment that could potentially lead to patient death. There are currently no available tools to observe how tumor cells communicate in vivo. Here, we developed two tools to visualize RNA-based cell-cell communication, using hybridization chain reactions (HCR) to kill cells that communicate and a Cre-lox gene construct to cause communicating cells to fluoresce. HCR that exhibited strong polymerization was based on hairpin 1 and 2 (H1/2) loop, stem, toe and overall length, as well as trigger oligonucleotide concentration. However, transfection with HCR components including and excluding a trigger oligonucleotide induced cell death. Plasmids containing targeted HCR component sequences flanked by a 5’-hammerhead ribozyme (HHR) and a 3’-Hepatitis Delta Virus (HDV) ribozyme were created to enable endogenous production of HCR components in cells. In vitro transcription of plasmids indicated ribozyme cleavage and release of target RNA. The H1, H2, and I RNA produced after transcribing the individual plasmids yielded HCR. Plasmids containing Cre recombinase, ribozyme, LoxP-STOP-LoxP, and green fluorescent protein (GFP) were created to permanently record cell-cell communication. The original gene construct with the start codon (ATG) in front of GFP caused strong false positive fluorescence in cells, while repositioning the ATG in front of LoxP-STOP-LoxP showed a dramatic decrease in fluorescence. To further reduce background fluorescence, a new plasmid was built with HHR at both the 5’- and 3’-end of Cre to ensure Cre gets cleaved and not made into a protein. Inhibitors complementary to the stemloops of each HHR were predicted in silico and tested, resulting in one candidate displaying ribozyme cleavage inhibition. Our studies demonstrate the ability of the HCR mechanism to mediate cell death in cultured human cancer cells. We further developed a Cre recombinase gene construct flanked by two ribozymes that can be specifically inhibited using a short trigger oligonucleotide. Both technologies show promise to become critical tools for visualizing RNA-based cell-cell communication and will be developed further in the future.
Description
This thesis has been embargoed for 5 years and will not be available until December 2020.
Keywords
Cell-cell communication, Hybridization chain reaction, RNA-based, Cell signaling