Immobilization-Free Determination of Dissociation Constants Independent of Ligand Size Using MicroScale Thermophoresis

Wiebke Sabrowski; Walter F M Stöcklein; Marcus M Menger

doi:10.1007/978-1-0716-2695-5_10

Immobilization-Free Determination of Dissociation Constants Independent of Ligand Size Using MicroScale Thermophoresis

Methods Mol Biol. 2023:2570:129-140. doi: 10.1007/978-1-0716-2695-5_10.

Authors

Wiebke Sabrowski^{1

2}, Walter F M Stöcklein¹, Marcus M Menger³

Affiliations

¹ Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalysis and Bioprocesses (IZI-BB), Potsdam, Germany.
² Institute of Chemistry and Biochemistry - Biochemistry, Freie Universität Berlin, Berlin, Germany.
³ Branch Bioanalysis and Bioprocesses, Fraunhofer Institute for Cell Therapy and Immunology, Potsdam, Germany. marcus.menger@izi-bb.fraunhofer.de.

PMID: 36156779
DOI: 10.1007/978-1-0716-2695-5_10

Abstract

The quantitative characterization of aptamer-ligand interactions is an important step in the aptamer development process. However, certain pitfalls impede K_D determination, especially when working with small molecule ligands. These include altered binding behavior caused by ligand immobilization. Further, the compulsory requirement for major differences in size between the bound and unbound state makes small molecule ligands ineligible for separation-based methods. MicroScale Thermophoresis circumvents such limitations as binding is accurately quantified with both binding partners free in solution and independent of ligand size. In this chapter, we present a protocol for the characterization of a DNA aptamer binding to its small molecule ligand daunorubicin.

Keywords: Aptamer; Aptamer-ligand interaction; Binding assay; Dissociation constant; Immobilization-free; MST; MicroScale Thermophoresis; Small molecules.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aptamers, Nucleotide* / chemistry
Daunorubicin
Ligands
Protein Binding

Substances

Aptamers, Nucleotide
Ligands
Daunorubicin