Aptamer Binding Studies Using MicroScale Thermophoresis

Methods Mol Biol. 2016;1380:99-111. doi: 10.1007/978-1-4939-3197-2_8.


The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer-target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.

Keywords: Aptamer–target interactions; Binding assay; Dissociation constant; Interaction affinity; MicroScale; Thermophoresis.

MeSH terms

  • Aptamers, Nucleotide* / metabolism
  • Humans
  • Protein Binding
  • SELEX Aptamer Technique / methods*
  • Thrombin / metabolism


  • Aptamers, Nucleotide
  • Thrombin