Cell surface RNA (csRNA) is a recent discovery in the field of RNA biology and has been implicated in playing important roles in many biological processes due to its extracellular properties. To understand the biogenesis, regulation, and function of csRNA, it is critical to develop methods to detect, isolate, and confidently characterize membrane-bound csRNA. Previously, csRNA has been profiled using methods based on cell membrane isolation that are expensive, laborious, and with unsatisfactory specificity and sensitivity . In this study, we use metabolic labeling and chemical cross-linking techniques to specifically label csRNA with biotin handles. We intended to use this technique for separating biotin-labeled csRNA from total RNA samples for characterization purposes. The primary materials that were used to label such csRNAs are 4-Thiouridine (4sU), an unnatural nucleotide analogue, and S-(2-aminoethyl)-ester-methanesulfonothioic-acid-biotin (MTSEA-biotin), a crosslinker designed specifically to label 4sU. By deploying these tools to cell lines such as HEK293T and HeLa, csRNA is detectable by Enhanced Chemiluminescent detection via Dot Blot. Furthermore, to separate biotin-labeled csRNA from total RNA, streptavidin-coated magnetic bead separation procedures could be used as a promising method for purifying csRNA from total RNA, for RNAseq characterization. This study highlights the processes of establishing the csRNA detection protocol and describes the current status and issues with developing the streptavidin-coated magnetic beads separation method. / Master of Science in Life Sciences / The 'central dogma' is a term that describes the process of DNA (a template-like molecule that holds all genetic coding within cells) transcribing into mRNA (a messenger molecule that transports this message to the ribosome) which is then translated into proteins (large, complex molecular machinery that is responsible for many biochemical functions within the body). However, RNA has been found to have a much wider range of functions than just being an intermediate messenger between DNA and proteins. Recently, short snippets of single nucleotide RNA strands have been discovered to be present on the outer cell membrane of certain mammalian cell types. The function of cell surface RNA (csRNA) is largely undiscovered, however, csRNA are likely involved in cell-cell interactions similar to outer membrane proteins, lipids, and carbohydrates. Currently, methods involved in detecting and characterizing csRNA are laborious, time extensive, and with unsatisfactory specificity and sensitivity. This study aims to develop novel methods to detect csRNA on different cell types in an undemanding and trustworthy manner to speed up research timelines while maintaining high confidence in results. Our design is to use metabolic labeling and click-chemistry to 'label' the csRNA. In this study, we describe early signs of detecting csRNA and how this was achieved. Additionally, the current status for separating and profiling csRNA sequences is discussed.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/119227 |
| Date | 03 June 2024 |
| Creators | Brooks, Maxwell David |
| Contributors | Biochemistry, Sun, Wei, Sobrado, Pablo, Lowell, Andrew Nesemann |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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