title: Aptamer Binding Studies Using MicroScale Thermophoresis.
abstract: 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.
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Breitsprecher
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Schlinck
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Witte
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Duhr
authors.affiliation: NanoTemper Technologies GmbH, Flößergasse 4, 81369, Munich, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Baaske
authors.affiliation: 2bind GmbH, Josef Engert Strasse 13, 93053, Regensburg, Germany. schubert@2bind.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schubert
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D052156:SELEX Aptamer Technique / Q000379:methods*; D013917:Thrombin / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D013917:Thrombin
keywords: Aptamer–target interactions; Binding assay; Dissociation constant; Interaction affinity; MicroScale; Thermophoresis