Thiazole orange-induced c-di-GMP quadruplex formation facilitates a simple fluorescent detection of this ubiquitous biofilm regulating molecule

J Am Chem Soc. 2011 Apr 6;133(13):4856-64. doi: 10.1021/ja1091062. Epub 2011 Mar 8.


Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have been shown to regulate bacterial virulence and biofilm formation. c-di-GMP can exist in different aggregate forms, and it has been demonstrated that the polymorphism of c-di-GMP is influenced by the nature of cation that is present in solution. In previous work, polymorphism of c-di-GMP could only be demonstrated at hundreds of micromolar concentrations of the dinucleotide, and it has been a matter of debate if polymorphism of c-di-GMP exists under in vivo conditions. In this Article, we demonstrate that c-di-GMP can form G-quadruplexes at low micromolar concentrations when aromatic molecules such as thiazole orange template the quadruplex formation. We then use this property of aromatic molecule-induced G-quadruplex formation of c-di-GMP to design a thiazole orange-based fluorescent detection of this important signaling molecule. We determine, using this thiazole orange assay on a crude bacterial cell lysate, that WspR D70E (a constitutively activated diguanylate cyclase) is functional in vivo when overexpressed in E. Coli . The intracellular concentration of c-di-GMP in an E. Coli cell that is overexpressed with WspR D70E is very high and can reach 2.92 mM.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Benzothiazoles / chemistry*
  • Biofilms*
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / analysis
  • Cyclic GMP / chemical synthesis
  • Cyclic GMP / chemistry
  • Escherichia coli / chemistry
  • Escherichia coli / cytology
  • Escherichia coli / metabolism
  • Fluorescence*
  • G-Quadruplexes*
  • Molecular Conformation
  • Quinolines / chemistry*


  • Benzothiazoles
  • Quinolines
  • thiazole orange
  • bis(3',5')-cyclic diguanylic acid
  • Cyclic GMP