Recent developments in the field of RNA biology continue to demonstrate the importance of RNA in regulating cellular processes. However, directly imaging biologically important RNAs has been hindered by a lack of live cell fluorescent tools. As such, aptamers that bind and enhance the brightness of fluorogenic dyes are promising tools to improve fluorescent RNA imaging. The Unrau laboratory developed RNA Mango I, a small, 39-nt aptamer that binds to a modified thiazole orange fluorophore (TO1-Biotin) with nanomolar affinity. This binding is accompanied by an 1,100-fold increase in its green channel fluorescence. To further improve the Mango aptamers, in collaboration with the Ryckelynck laboratory, we used microfluidics-based selection methods to isolate three brighter, high affinity RNA Mango fluorogenic aptamers (Mango II, III and IV). Together with the Rueda laboratory, we show that these new Mangos can accurately image the sub-cellular localization of three small non-coding RNAs in fixed and live mammalian cells. These new Mangos are unique in structure. Unlike Mango I and II, Mango III rigidly connects its ligand binding core to an external helix. As the Spinach/Broccoli aptamer family, which binds GFP-like chromophores (DFHBI, DFHBI-1T), also share this property, the Broccoli/DFHBI-1T aptamer complex was used as a FRET donor paired with the far red-shifted Mango III/YO3-Biotin complex as a FRET acceptor. Interestingly, the high affinity Mango I, II, III aptamers can discriminate between TO1/YO3-Biotin and DFHBI/DFHBI-1T by at least a 102-fold difference in affinity. In contrast, Spinach binds many fluorophores indiscriminately and weaker. With this, concentrations could be determined to obtain appropriate binding for Mango III/YO3-Biotin and Broccoli/DFHBI-1T when in the same system. FRET efficiency was measured using an RNA duplex of variable length between the two aptamers. FRET signal depended on the length of the duplex, and oscillated in intensity precisely with the predicted twist of the helix, demonstrating strong orientation dependence. While this pair of aptamers enable in vitro FRET studies, there are no truly orthogonal fluorescent aptamer systems. To that end, I discuss an in vitro selection to develop orthogonal aptamers for a red fluorophore, TO3-Biotin, that can potentially be paired with existing Mango aptamers.
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Thesis advisor: Unrau, Peter
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