| dc.description.abstract | Aptamers are short single-stranded DNA or RNA sequences that have been selected in vitro to have a high binding affinity to a specific target molecule. One class of aptamers can bind to small non-fluorescent molecules to result in a dramatic fluorescence enhancement, once bound. These aptamer sequences, along with their cognate fluorogenic molecules, are used as light-up probes. RNA light-up probes are used to monitor sub-cellular localization of RNA in cell with the RNA aptamer Mango I being notable for having a high affinity along with excellent fluorescence enhancement. The usage of RNA aptamers in biosensing application is limited due to the fact that the degradation of RNA due to the pervasive RNase is a key factor in the sensors shelf life. For this reason, DNA aptamers are the choice for usage in biosensing applications.
In chapter 2, a new DNA aptamer, named 02a, that binds to the target TO1-biotin was isolated after nine rounds of Systematic Evolution of Ligands by EXponential enrichment (SELEX). The selection was performed on streptavidin coated beads with the target bound to the surface with free dye in solution in higher selection rounds to select for slower off-rate binding. Using Next Generation Sequencing (NGS), the library after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. The 02a sequence family emerged showing superior fluorescence enhancement, 136.5 times more than unbound TO1-biotin, as well as high affinity, with a KD of 98.7 nM, for the target, when compared to other families obtained by NGS analysis.
In chapter 3, several sequence families were studied to discern structural information to better understand the binding interaction. Using several primary and secondary sequence analysis tools, a three-tiered G-quadruplex motif for the families was predicted. For the 02a aptamer, it was found that binding and fluorescence enhancement was attenuated in the absence of K+ and that the region outside the predicted quadruplex gives access to potassium-dependent fluorescence enhancement conformers.
In chapter 4, biosensing approaches were evaluated for their use in the detection of the biomarker IgE. An aptamer for IgE, D17.4, was used in several sensing platforms such as Localized Surface Plasmon Resonance (LSPR), Electrophoretic Mobility Shift Assay (EMSA), Förster Resonance Energy Transfer (FRET), and Lateral Flow ImmunoAssay (LFIA) as well as in a modular aptamer configuration and was used to evaluate the detection of isolated IgE and IgE in human serum. Although the detection of IgE was possible in most set-ups, complex optimization steps are needed for optimization for commercial use, especially when using human serum samples.
Chapter 5 summarizes all of the findings and future work is discussed. Overall, in vitro selection was used to select for a DNA aptamer for the thiazole orange derivative TO1-biotin which was then characterized to have a quadruplex motif. This novel aptamer, 02a, is a useful fluorescent module for biosensing applications and has superior characteristics than other published light-up DNA aptamers | en |
