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Updated: Apr 28, 2026
A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay
Published on: February 28, 2015
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.
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.
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.
