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Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis
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High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn

Kylan Szeto1, Sarah J Reinholt, Fabiana M Duarte

  • 1School of Applied and Engineering Physics, Cornell University, Ithaca, NY, 14853, USA.

Analytical and Bioanalytical Chemistry
|February 21, 2014
PubMed
Summary

A new 96-well device enables high-throughput aptamer selection using affinity microcolumn chromatography. This versatile system significantly reduces time and cost for aptamer discovery and characterization.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Aptamer selection is crucial for developing molecular tools.
  • Current methods can be time-consuming and costly.
  • Plate-based processing offers potential for automation.

Purpose of the Study:

  • To introduce a versatile 96-well microplate device for aptamer selection.
  • To demonstrate its utility in high-throughput characterization of aptamer-target binding.
  • To reduce time and cost in aptamer discovery.

Main Methods:

  • Developed a reconfigurable 96-microcolumn device for affinity chromatography.
  • Operated the device in serial and parallel modes for aptamer selections.
  • Performed 96 selection tests to characterize aptamer binding to protein targets.

Main Results:

  • Demonstrated significant aptamer enrichment on specific protein targets (hHSF1, NELF-E, GFP).
  • Achieved high-fold enrichments: 200-fold for hHSF1/NELF-E, 750-fold for GFP.
  • Confirmed aptamer specificity, with minimal binding to non-target molecules.
  • Identified optimal target concentrations for aptamer binding, consistent with steric hindrance.

Conclusions:

  • The 96-well microplate device facilitates efficient, high-throughput aptamer selection and characterization.
  • Integration with plate-based systems streamlines downstream processes.
  • This approach significantly reduces the resources required for aptamer discovery.