Phosphate-Modified Nucleotides for Monitoring Enzyme Activity

  • Susanne Ermert
  • Andreas Marx
  • Stephan M. HackerEmail author
Part of the following topical collections:
  1. Phosphate Labeling and Sensing in Chemical Biology


Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.


Modified nucleotides Activity assays Kinases DNA sequencing Fluorogenic substrates FRET 



Financial support by the Deutsche Forschungsgemeinschaft (Grant SFB 969) and the Konstanz Research School Chemical Biology is gratefully acknowledged. S.M.H. also acknowledges the Deutsche Forschungsgemeinschaft, the Studienstiftung des deutschen Volkes and the Zukunftskolleg of the University of Konstanz for stipends.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there is no conflict of interest regarding this publication.


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Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Susanne Ermert
    • 1
  • Andreas Marx
    • 1
  • Stephan M. Hacker
    • 1
    Email author
  1. 1.Department of Chemistry and Konstanz Research School Chemical BiologyUniversity of KonstanzKonstanzGermany

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