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Quantitative Characterization of Partitioning Stringency in SELEX.

An T H Le1,2, Eden Teclemichael1,2, Svetlana M Krylova1,2

  • 1Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.

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Controlling stringency in Systematic Evolution of Ligands by EXponential enrichment (SELEX) is vital. This study introduces the binder-to-nonbinder ratio (BNR) to quantitatively measure stringency, preventing SELEX failure.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Aptamer selection via SELEX requires stringent conditions for high-affinity binders.
  • Excessive stringency in SELEX increases the risk of selection failure.
  • Current SELEX stringency control relies on intuition due to a lack of quantitative measures.

Purpose of the Study:

  • To develop a quantitative measure for SELEX stringency.
  • To establish a criterion for preventing SELEX failure.
  • To enable more rational control over aptamer selection processes.

Main Methods:

  • Quantified stringency using the binder-to-nonbinder ratio (BNR).
  • Developed an experimental method to determine BNR via quantitative PCR.
  • Analyzed theoretical aspects and conducted SELEX experiments with two protein targets.

Main Results:

  • Introduced BNR as a quantitative measure of SELEX stringency, where lower BNR indicates higher stringency.
  • Established the SELEX nonfailure criterion: maintaining a BNR significantly greater than zero.
  • Demonstrated that inverse BNR can be used to quantify stringency and guide SELEX progress.

Conclusions:

  • The BNR provides a rational approach to controlling SELEX stringency and avoiding failure.
  • The SELEX nonfailure criterion and BNR are applicable to other artificial evolution methods.
  • Quantitative stringency measurement enhances the reliability and success rate of aptamer selection.