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Fluorescent 2-Aminopurine c-di-GMP and GpG Analogs as PDE Probes

  • Jie Zhou
  • Clement Opoku-Temeng
  • Herman O. SintimEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1657)

Abstract

c-di-GMP is widely recognized as an important ubiquitous signaling molecule in bacteria. c-di-GMP phosphodiesterases (PDEs) regulate the intracellular concentration of c-di-GMP and some could be potential drug targets. Here, we describe a class of dinucleotide probes suitable for monitoring the enzymatic activities of c-di-GMP PDEs in real time. Such probes contain fluorescent nucleobases and can be readily cleaved by PDEs, resulting in a change in fluorescence. Fluorescent cyclic and linear dinucleotide probes could be used in diverse applications, such as confirming the activity of an expressed PDE or oligoribonuclease (Orns) or identifying inhibitors of PDEs or Orns using high-throughput screening formats.

Key words

c-di-GMP Fluorescent bases c-di-GMP synthases c-di-GMP PDEs 2-Aminopurine (2AP) Oligoribonuclease (Orn) High-throughput screening Inhibitor 

Notes

Acknowledgment

This work was funded by the National Science Foundation (CHE1307218 and CHE1636752), Purdue University. Plasmids were provided by Dr. Zhaoxun Liang (RocR plasmid), Dr. Ehud Banin (P. aeruginosa Orn plasmid), and Dr. Nicholas Dixon (E. coli and M. smegmatis Orn plasmids).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jie Zhou
    • 1
    • 2
  • Clement Opoku-Temeng
    • 1
    • 5
    • 3
  • Herman O. Sintim
    • 1
    • 2
    • 4
    Email author
  1. 1.Purdue Institute for Drug DiscoveryPurdue UniversityWest LafayetteUSA
  2. 2.Department of ChemistryPurdue UniversityWest LafayetteUSA
  3. 3.Biochemistry Graduate ProgramUniversity of MarylandCollege ParkUSA
  4. 4.Purdue Institute of Inflammation, Immunology and Infectious DiseaseWest LafayetteUSA
  5. 5.Department of Chemistry, Center for Drug DiscoveryPurdue UniversityWest LafayetteUSA

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