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Abstract
Aptamers are DNA or RNA oligonucleotide-based bioreceptors isolated in vitro through the Systematic Evolution of Ligands by Exponential Enrichment. Given the ease with which a selection can be customized, aptamers can be evolved to function in nearly any chemical environment, making them tailormade for their final application. However, the post-SELEX characterization of the 100-1000’s aptamer candidates remains a significant bottleneck as there are no suitable techniques for high-throughput characterization of each candidate’s affinity/specificity. Moreover, the final aptamer must be engineered to possess signal reporting functionality; this is often done via trail-and-error truncation to yield a structure-switching aptamer. This dissertation describes the development of an exonuclease-based fluorescence assay that can simultaneously engineer structure-switching aptamers from their parent aptamers and provide the binding profile of these truncated aptamers. We first demonstrate that a mixture of Exonuclease III (Exo III) and Exonuclease I (Exo I) could detect small-molecule target-binding events in fully folded aptamers yielding a truncated intact oligonucleotide product in the presence of the target, but completely digests unbound aptamers into mononucleotides. We utilized this phenomenon to construct a highly sensitive enzyme-assisted aptamer-based sensor using SYBR Gold dye to report the presence of the inhibition product as a proxy for target concentration in biological matrixes or molecular beacons for multiplexed detection of small-molecule targets simultaneously in a single reaction volume. We then used a panel of aptamer mutants to demonstrate a qualitative relationship between target-induced enzymatic inhibition and a mutant’s binding affinity. This was further confirmed as a qualitative relationship using a testbed of 28 newly isolated aptamers for 655 aptamer-ligand pairs. Characterization of the inhibition products observed during these tests revealed that it possesses structure-switching functionality, and the truncated products can be incorporated into electrochemical aptamer-based (E-AB) sensors. Finally, we applied our assay to generate a truncated THC-binding aptamer, which was then incorporated into an E-AB sensor to detect THC in the plant extract. The work done in this dissertation highlights the strength of the exonuclease-based fluorescence assay for aptamer characterization, engineering, and sensor development.