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

Aptamer Binding Studies Using MicroScale Thermophoresis.

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NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
2bind GmbH, Josef Engert Strasse 13, 93053, Regensburg, Germany.


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.


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

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