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Author ORCID Identifier

https://orcid.org/0000-0003-3875-9111

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2022

Month Degree Awarded

February

First Advisor

Mingxu You

Subject Categories

Analytical Chemistry | Biochemistry | Biotechnology | Cell Biology | Molecular Biology

Abstract

Fluorescence imaging of intracellular small molecules such as metabolites and signaling molecules is crucial for understanding various biological pathways and disease related phenomena. In depth understanding of metabolism or signal transducing pathways could provide valuable insights in biomedical field. Organic dye probes were previously used as reporters to image various target molecules. These probes may raise issues such as high toxicity and nonspecific interaction with cellular components, etc., which highly limited their application in various purposes. The development of genetically encoded fluorescent protein-based probe has revolutionized the bioimaging field. Fluorescent protein-based sensors exhibit high biocompatibility and show high specificity towards their target molecules. However, the limited signal-to-noise ratio and the lack of recognition units that can be used to develop sensors have hindered the application of these probes in imaging many cellular targets of interest. By mimicking green fluorescent proteins, a type of fluorogenic RNA molecule has recently emerged and started to revolutionize the development of biosensors for the intracellular imaging of various target analytes, especially small molecules and nucleic acids. The versatile design strategy and the possibility of identifying RNA aptamers for almost all small-molecule targets via high-throughput directed evolution method have made these fluorogenic RNA-based sensors become more and more popular in use of cellular imaging and biomedical research. Even though these fluorogenic RNA-based sensors have been used to detect various targets both in vitro and in cellular environment, there are still questions and challenges that need to be addressed such as accurate quantification of analytes, detection of multiple targets at the same time and potential point-of-care application of RNA sensors.

First, to accurately determine the intracellular concentration of target analytes to better understand the biological pathways, we designed a ratiometric imaging system by using two pairs of orthogonal fluorogenic RNA aptamer/dye: Broccoli/DFHBI-1T and DNB/SR-DN. Herein, Broccoli was used as an internal reference to normalize the RNA expression level in each individual cell. DNB was developed as the first red color emitting modular sensor that can be engineered to detect a variety of target molecules. As a result, this ratiometric sensor platform was able to accurately quantify the amount of antibiotic, tetracycline and another signaling molecule, c-di-GMP at the single-cell level. Second, to monitor the dynamics of targets in living cells to better understand the metabolism and signaling pathways, we used a more photostable dye, TMR-DN to replace SR-DN dye. The new pairs of fluorogenic RNA aptamer/dye, Broccoli/DFHBI-1T and DNB/TMR-DN, could still maintain the quantification capability and have been successfully used to monitor tetracycline dynamics in bacterial cells. Third, with the increasing issues arising from antibiotic resistance especially from the clinically relevant antibiotics, we aim to develop RNA sensors for gentamicin, one of the widely used antibiotics in clinics. The development of gentamicin targeting RNA sensors would help us to understand gentamicin resistance and to screen efflux pump inhibitors to elevate the intracellular concentration of gentamicin to improve the treating efficiency. Unfortunately, there are no existing RNA aptamers that can tightly and specifically bind with gentamicin. To achieve RNA aptamers, we used a systematic evolution of ligands by exponential enrichment (SELEX) strategy to discover gentamicin-binding RNA aptamers, which can be further engineered for the development of gentamicin-targeting fluorescent RNA sensors. After performing 13 rounds of selection, several potential aptamer sequences were identified through next generation sequencing. While these strands need further characterization to validate their capability of binding with gentamicin, the development of these gentamicin-targeting RNA sensors could potentially be applied to identify novel efflux pump inhibitors, which can be used in a combinational therapy to treat patients who are suffering from infectious diseases. Fourth, for applying RNA sensors in multiplex imaging to better understand biological processes that involve the participation of a group of biomolecules, we developed a fluorogenic RNA-based multiplex imaging system using multiple cycles of image-wash. We identified four pairs of orthogonal fluorogenic RNA aptamer/dye and have demonstrated the simultaneous imaging of four RNAs using three cycles of image-wash. We further utilize this multiplex imaging system to determine the dynamics of three targets of interest including c-di-GMP, ppGpp and SAM under different treatment such as antibiotics, nutrient starvation and biofilm formation. This novel RNA-based multiplex imaging system could be potentially used to study complex cellular processes that involve multiple biomolecules. Finally, to demonstrate the potential application of RNA sensors in point-of-care detection, we incorporated fluorogenic RNA-based sensors into a paper substrate for the first time. Broccoli-based sensors, DFHBI-1T and buffer were embedded in the cellulose matrix to achieve long-term storage. Upon addition of target analytes, the Broccoli fluorescence can be activated specifically and rapidly. Paper-based tetracycline and ppGpp sensors have been developed and exhibit optimal sensor performance after storing at room temperature for a certain period.

DOI

https://doi.org/10.7275/26819405.0

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Wednesday, February 01, 2023

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