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

• NCJ Number: 308423
• Author(s): Juan Canoura
• Date Published: 2022
• Grant Number(s): 2019-R2-CX-0045
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)

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.