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Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

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A new method using exonucleases rapidly assesses aptamer binding affinity, improving aptamer engineering. This technique enhances aptamer selection for specific targets like adenosine, enabling sensitive biosensor development.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is used to isolate aptamers, but identifying optimal sequences with high affinity and specificity is challenging.
  • Existing post-SELEX aptamer engineering methods often have limitations, including bias, variable success rates, and the need for specialized equipment.

Purpose of the Study:

  • To develop a generalizable, rapid, label-free assay for interrogating the binding properties of aptamers.
  • To engineer aptamers with improved binding characteristics, specifically enhancing affinity and specificity for target molecules.
  • To demonstrate the applicability of the developed method for both small-molecule and protein-binding aptamers.

Main Methods:

  • Utilized exonuclease III and exonuclease I to analyze binding properties of aptamers by monitoring changes in digestion kinetics upon ligand binding.
  • Applied the assay to an ochratoxin-binding DNA aptamer and its mutants to correlate ligand binding with exonuclease digestion kinetics.
  • Engineered a DNA aptamer with indiscriminate binding to ATP, ADP, AMP, and adenosine by screening mutants and identifying high-affinity adenosine-specific aptamers.

Main Results:

  • Ligand binding altered exonuclease digestion kinetics in a manner that closely correlated with aptamer-ligand affinity.
  • Identified two high-affinity aptamers that specifically bind to adenosine from a library of mutants.
  • Developed an electrochemical aptamer-based sensor using the engineered aptamers, achieving a 1 μM detection limit for adenosine in 50% serum.

Conclusions:

  • The developed exonuclease-based assay is a generalizable and effective method for characterizing aptamer-ligand binding affinities.
  • This approach facilitates the engineering of aptamers with improved binding properties, suitable for various applications, including biosensing.
  • The method is adaptable for high-throughput screening and can be applied to DNA aptamers of diverse sequences, structures, and lengths.