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Exonuclease Digestion Assay for Streamlining Aptamer Characterization, Engineering, and Sensor Development

NCJ Number
308423
Author(s)
Juan Canoura
Date Published
2022
Annotation

In this paper, the authors highlight the adaptability of aptamers and the strength of the authors’ exonuclease-based fluorescence assay for aptamer characterization, engineering, and sensor development.

Abstract

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 the truncated aptamers. The authors first demonstrate that while a mixture of Exonuclease III (Exo III) and Exonuclease I (Exo I) can detect small-molecule target-binding events in fully folded aptamers yielding a truncated intact oligonucleotide product in the presence of the target, it completely digests unbound aptamers into mononucleotides. They then describe using a panel of aptamer mutants to demonstrate a qualitative relationship between target-induced enzymatic inhibition and a mutant’s binding affinity; the authors confirmed this as a qualitative relationship using a testbed of 28 newly isolated aptamers for 655 aptamer-ligand pairs. They note that characterization of the inhibition products observed during those tests revealed that it possesses structure-switching functionality, and the truncated products can be incorporated into electrochemical aptamer-based (E-AB) sensors. Finally, they report applying their 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 paper demonstrates the strength of the exonuclease-based fluorescence assay for aptamer characterization, engineering, and sensor development.