FRET-based Optical Assay for Monitoring Riboswitch Activation

Biomacromolecules. 2009 May 11;10(5):1055-60. doi: 10.1021/bm801117f.

Abstract

Riboswitches are regulatory RNAs located in the 5'-untranslated region of mRNA sequences that recognize and bind to small molecules and regulate the expression of downstream genes. Creation of synthetic riboswitches to novel ligands depends on the ability to monitor riboswitch activation in the presence of analyte. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically encoded fluorescent proteins. The theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence. Our FRET construct was composed of eGFP and a nonfluorescent yellow fluorescent protein mutant called REACh (for resonance energy-accepting chromoprotein) connected with a peptide linker containing a TEV protease cleavage site. Addition of theophylline to the E. coli cells activates the riboswitch and initiates the translation of mRNA. Synthesized protease cleaves the linker in the FRET-based fusion protein causing a change in the fluorescence signal. By this method, we observed an 11-fold increase in cellular extract fluorescence in the presence of theophylline. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence. This leads to an improved detection of FRET via better signal-to-noise ratio, allowing us to monitor riboswitch activation in a wide range of analyte concentrations from 0.01 to 2.5 mM.

Publication types

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

MeSH terms

  • Endopeptidases / chemistry
  • Endopeptidases / metabolism
  • Escherichia coli / cytology
  • Escherichia coli / metabolism
  • Fluorescence
  • Fluorescence Resonance Energy Transfer*
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / metabolism
  • Kinetics
  • Ligands
  • RNA / chemistry
  • RNA / genetics
  • RNA / metabolism*
  • Theophylline / chemistry
  • Theophylline / metabolism

Substances

  • Ligands
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • RNA
  • Theophylline
  • Endopeptidases
  • TEV protease