Doctor of Philosophy (PhD)
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Aptamer, in vitro Selection, SELEX, small molecule detection, exonuclease
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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.
Previously Published In
J. Am. Chem. Soc. 2021, 143, 2, 805–816
J. Am. Chem. Soc. 2018, 140, 31, 9961–9971
Canoura, Juan, "Exonuclease Digestion Assay for Streamlining Aptamer Characterization, Engineering, and Sensor Development" (2022). FIU Electronic Theses and Dissertations. 5023.
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