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A simple method for eliminating fixed-region interference of aptamer binding during SELEX.

Eric Ouellet1, Eric T Lagally, Karen C Cheung

  • 1Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z4; Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3; Biomedical Engineering Program, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3.

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Summary

Flanking sequences in aptamer selection libraries can interfere with aptamer folding and function. This study introduces a complementary oligonucleotide method to block this interference, restoring aptamer binding affinity and improving selection efficiency.

Keywords:
DNA aptamerRNA aptamerSELEXaptamernucleic acidspolymerase chain reactiontherapeutics

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biochemistry

Background:

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a key method for aptamer discovery.
  • Standard SELEX libraries contain flanking sequences that can unintentionally bias selection outcomes.
  • These flanking sequences may interfere with aptamer structure and binding function.

Purpose of the Study:

  • To investigate and mitigate the interference caused by flanking sequences in SELEX libraries.
  • To develop a method for blocking flanking sequence interference and restoring aptamer binding affinity.
  • To enhance the efficiency of aptamer selection and enrichment.

Main Methods:

  • Utilized isothermal titration calorimetry (ITC) to analyze interference mechanisms and assess blocking efficacy.
  • Employed fold predictions to understand aptamer-flanking sequence interactions.
  • Applied a complementary oligonucleotide blocking strategy to benchmark aptamer libraries (thrombin, streptavidin, VEGF).
  • Developed a qPCR-based method for determining mean binding affinity of aptamer pools.

Main Results:

  • Complementary oligonucleotide blocking effectively eliminated fixed-region mediated interference.
  • Natural binding affinities of benchmark aptamers were fully restored after blocking.
  • The blocking protocol did not affect the binding affinities of functional aptamers.
  • The method demonstrated successful application across various library formats and flanking sequences.
  • A rapid qPCR assay was established for aptamer pool binding affinity assessment.
  • Pre-blocking the library significantly improved the retention of high-affinity aptamers in early selection rounds.

Conclusions:

  • Blocking flanking sequence interference is crucial for accurate aptamer selection.
  • The complementary oligonucleotide method offers a robust strategy to enhance SELEX efficiency.
  • This technique isolates aptamer sequence and function within the random region, complementing existing SELEX protocols.