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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
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High-Throughput Aptamer Characterization via Real-Time Nuclease Digestion.

Caleb Byrd1, Obtin Alkhamis1, Gabriel Castro1

  • 1Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Raleigh, North Carolina 27695, United States.

Journal of the American Chemical Society
|March 2, 2026
PubMed
Summary
This summary is machine-generated.

A new real-time exonuclease (RT-Exo) assay streamlines aptamer characterization. This high-throughput method accurately screens aptamer-ligand interactions, significantly improving efficiency for diverse applications.

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Area of Science:

  • Biotechnology and Molecular Biology
  • Analytical Chemistry
  • Biochemistry

Background:

  • Aptamers are valuable biorecognition elements for various applications due to their high-affinity binding.
  • Characterizing aptamer-ligand interactions accurately and at high throughput remains a significant challenge.
  • Previous exonuclease digestion assays offered parallel, label-free screening but lacked sufficient throughput.

Purpose of the Study:

  • To develop a next-generation, high-throughput assay for screening aptamer-ligand interactions.
  • To improve the efficiency, precision, and simplicity of aptamer characterization.
  • To enable label-free, real-time monitoring of aptamer binding.

Main Methods:

  • Development of a real-time exonuclease (RT-Exo) assay utilizing a fluorogenic aptamer reporter.
  • Monitoring exonucleolytic digestion of aptamers in real time using automated readers.
  • Validation of the RT-Exo assay with diverse aptamers and targets under various conditions.

Main Results:

  • The RT-Exo assay achieves throughput orders of magnitude higher than previous methods with reduced labor and resources.
  • The assay demonstrated robust performance across different aptamer structures and target properties.
  • Successful application in screening mutants for structure-activity studies and profiling binding to 120 fentanyl analogs in a single run.

Conclusions:

  • The RT-Exo assay significantly enhances the throughput, precision, and simplicity of aptamer characterization.
  • This method streamlines the process of screening aptamer-ligand interactions.
  • The RT-Exo assay is a powerful tool for advancing aptamer-based applications and research.