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Author ORCID Identifier

https://orcid.org/0000-0001-9980-3011

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

September

First Advisor

Mingxu You

Subject Categories

Genetics | Genetics and Genomics | Microbiology | Molecular Biology | Molecular Genetics | Nanotechnology | Nucleic Acids, Nucleotides, and Nucleosides | Other Genetics and Genomics | Research Methods in Life Sciences

Abstract

Fluorescence imaging of cellular components and biomolecules have given numerous insights in biomedical research. After initial achievements with synthetic organic molecule-based imaging probes, protein-based imaging tools have led this field with their biocompatibility and high specificity. However, the limited choice of probes and low signal-to-noise ratio have started to slow down the pace of protein-aided fluorescence imaging. With the discovery of Spinach, a fluorogenic RNA aptamer mimicking green fluorescence protein, in 2011, genetically encoded RNA devices have become a game changer for intracellular fluorescence imaging and target detections. The general design strategy and broad target choice of these RNA-based sensors have let them gain more and more attentions for advanced imaging and research applications. In this thesis, we will use several examples to demonstrate how these genetically encoded RNA devices are able to perform advanced imaging in live cells.

First, in order to understand the antibacterial effects from silver ions and silver nanoparticles, we designed a so-called aptamer-free RNA sensor by inserting a cytosine-silver ion-cytosine metallo base pair into a Broccoli RNA aptamer for the silver ion detection. Both in vitro and intracellular results demonstrated the successful detection and imaging of silver ions using these genetically encoded RNA sensors. Second, with the more and more urgent issues from antibiotic resistance, we developed several antibiotic-targeting RNA sensors using general allosteric design principle, which can be used to image antibiotic levels inside live bacterial cells to help understand antibiotic resistance and screen for new efflux pump inhibitors. Third, for applying RNA sensors in deep tissue and animal studies, we demonstrated the first genetically encoded RNA bioluminescent sensors based on bioluminescence resonance energy transfer (BRET). By coupling a NanoLuc luciferase with fluorogenic RNA sensors, these RNA-based BRET sensor can be potentially applied for the real in vivo imaging. Finally, to solve the limitations within current DNA-PAINT probes, we initiated the development of genetically encoded fluorogenic RNA probes for the super-resolution imaging in live cells. We proposed a new fluorogenic RNA-based points accumulation for imaging in nanoscale topography (fRNA-PAINT) approach to achieve such advanced imaging.

DOI

https://doi.org/10.7275/24376575

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 Thursday, September 01, 2022

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