Date of Award
Master of Science
Knowledge about ribonucleic acids (RNAs) and their roles has recently been expanded by extensive studies seeking to uncover novel RNAs using biochemical techniques. These techniques visualize specific RNA in situ to understanding the regulatory mechanisms of gene expression. Lately, a variety of photo labeling techniques has been developed for imaging RNA in vivo to locate and track it with high accuracy. This study focuses on fluorescence-based methods of in vivo RNA visualization. Using fluorescent light has transformed the study of micro-molecular components due to its many advantages. Molecular beacons targeting repeat expansion of RNA can differentiate between normal repeat sizes and repeat expansions in the pathogenic range. With modification and testing, molecular beacons have the potential to serve as probes for sensing repeat structure accessibility and for use in diagnostics assays. Broccoli GFP-mimic RNAs have different instability and metal ion preferences than Baby Spinach, a truncated version of the Spinach aptamer, that are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs, which likely explains their sensitivity to magnesium due to its stability and fluorescence. RNA Mango is an RNA aptamer that binds thiazole Orange (TO1) dye and its derivative with nanomolar binding affinity, unlike MS2 and Spinach systems. Mango is superior in its detection of bacterial fluorescence compared with Spinach, which requires 200uM of the fluorophore. Inserting a Mango aptamer with TO1-Biotin, a derivative of TO1, genetically into the cell can be used to monitor the expression of RNA in real time by measuring the fluorescence signal that comes from the TO1-Biotin.
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