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Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

Juan Hu1, Joonyul Kim1, Christopher J Easley1

  • 1Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, AL 36849.

Analytical Methods : Advancing Methods and Applications
|September 15, 2015
PubMed
Summary
This summary is machine-generated.

A new thermofluorimetric analysis (TFA) method enables quantitative protein detection using aptamers. This assay offers a simpler, more generalizable readout for aptamer-protein binding, even in complex samples like human serum.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Diagnostics

Background:

  • Aptamer-based protein assays typically rely on optical or electrochemical readouts of DNA hybridization.
  • Aptamer-target binding mechanisms can interfere with traditional assay readouts, complicating method development.
  • A need exists for simpler, more generalizable methods to quantify aptamer-protein interactions.

Purpose of the Study:

  • To introduce thermofluorimetric analysis (TFA) as a quantitative readout for aptamer-protein binding.
  • To demonstrate the utility of TFA for protein quantification in complex biological matrices.
  • To establish a robust and generalizable method for aptamer-based protein assays.

Main Methods:

  • Utilized thermofluorimetric analysis (TFA) coupled with DNA intercalating dyes to monitor aptamer-protein binding.
  • Employed fluorescence melting analysis (-dF/dT curves) to thermally resolve bound and unbound aptamers.
  • Tested the method using platelet-derived growth factor (PDGF) and its corresponding aptamer in various matrices, including human serum.

Main Results:

  • Achieved sub-nanomolar detection of platelet-derived growth factor (PDGF), with a limit of detection of 0.74 nM.
  • Demonstrated homogeneous optical detection in human serum with PDGF detection limits of 1.8 nM and 10.7 nM at different dilutions.
  • Successfully resolved bound and unbound aptamers in a homogeneous format using standard qPCR instrumentation.

Conclusions:

  • Thermofluorimetric analysis (TFA) provides a simple, generalizable, and quantitative method for aptamer-based protein assays.
  • The TFA approach overcomes limitations of traditional hybridization-dependent readouts and is effective in complex biological samples.
  • This method represents a significant advancement towards robust and widely applicable aptamer-based protein detection systems.