Quantifying the Number and Affinity of Mn2+-Binding Sites with EPR Spectroscopy

Christine Wuebben; Olav Schiemann

doi:10.1007/978-1-0716-2047-2_7

Quantifying the Number and Affinity of Mn²⁺-Binding Sites with EPR Spectroscopy

Methods Mol Biol. 2022:2439:91-101. doi: 10.1007/978-1-0716-2047-2_7.

Authors

Christine Wuebben¹, Olav Schiemann²

Affiliations

¹ Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University, Bonn, Germany.
² Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University, Bonn, Germany. schiemann@pc.uni-bonn.de.

PMID: 35226317
DOI: 10.1007/978-1-0716-2047-2_7

Abstract

During the last decades, various functional oligonucleotides have been discovered including DNAzymes, ribozymes, and riboswitches. Their function is based on their ability to form and change their three-dimensional structure. Binding of divalent ions to specific binding pockets was found to be important for the global structure and function. Here, we present a protocol that allows counting the number of Mn²⁺-binding sites and to determine their dissociation constants by means of continuous wave X-band Electron Paramagnetic Resonance (EPR) spectroscopy. In this method, Mn²⁺ is titrated into the oligonucleotide-containing sample and the intensity of the EPR spectrum is recorded. By comparison with a Mn²⁺-only reference sample, the binding isotherm can be constructed and fitted to binding models yielding the number and affinities of the binding sites. This method has been successfully applied to several functional oligonucleotides.

Keywords: Binding isotherms; Binding sites; EPR spectroscopy; Metal ions; Mn2+ titration.

MeSH terms

Binding Sites
Electron Spin Resonance Spectroscopy / methods
Manganese* / chemistry
RNA, Catalytic* / metabolism

Substances

RNA, Catalytic
Manganese