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High-throughput quantitative binding analysis of DNA aptamers using exonucleases.

Juan Canoura1,2, Obtin Alkhamis1, Yingzhu Liu1

  • 1Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC 27607, USA.

Nucleic Acids Research
|December 30, 2022
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Summary

We developed a novel high-throughput method to quantify aptamer binding properties using exonuclease digestion kinetics. This accelerates aptamer selection for developing sensitive fentanyl sensors.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Aptamers are nucleic acid bioreceptors with applications in diagnostics and therapeutics.
  • Identifying optimal aptamers for specific applications is a significant challenge.
  • Current methods for aptamer characterization can be time-consuming and complex.

Purpose of the Study:

  • To develop a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity.
  • To demonstrate the utility of this method by isolating and characterizing aptamers for fentanyl and its analogs.
  • To streamline aptamer selection for the development of advanced sensors.

Main Methods:

  • Developed a novel assay based on the kinetics of aptamer digestion by exonucleases to quantify binding properties.
  • Isolated new aptamers targeting fentanyl and its analogs.
  • Characterized 655 aptamer-ligand pairs using the exonuclease digestion assay.
  • Validated results with gold-standard methodologies.

Main Results:

  • Successfully developed and validated a high-throughput method for aptamer characterization.
  • Isolated novel aptamers with characterized binding properties for fentanyl detection.
  • Demonstrated the ability to select optimal aptamers for sensor development.
  • The assay significantly accelerates the aptamer characterization process.

Conclusions:

  • The exonuclease digestion kinetics assay provides a rapid and accurate method for aptamer characterization.
  • This approach facilitates the development of highly specific and sensitive aptamer-based sensors.
  • The method has the potential for further high-throughput analysis with robotic integration.