Dimerization-based homogeneous fluorosensor proteins for the detection of specific dsDNA

Nucleic Acids Symp Ser (Oxf). 2007:(51):307-8. doi: 10.1093/nass/nrm154.

Abstract

While there are many hybridization-based DNA sensors, few can detect native double-stranded DNA (dsDNA), which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers with an N-terminal dimerization motif and a C-terminal GFP variant to detect specific dsDNA sequence in a homogeneous solution. When a pair of purified zinc finger-GFP colour variant proteins (Zif12-eCFP, Zif12-eYFP) were mixed and added with specific dsDNA with 12 bp inverted repeat sequence, fluorescence spectra of the solution showed significant concentration-dependent enhancement of fluorescence resonance energy transfer (FRET), with the detection limit of approximately 10 nM. No significant change in FRET was observed upon addition of dsDNA with non-specific sequence, indicating dsDNA-dependent dimerization of the two proteins. This dimerization-based dsDNA sensors will have a range of applications where conventional hybridization-based assay is difficult.

MeSH terms

  • DNA / chemistry
  • DNA-Binding Proteins / chemistry*
  • Dimerization
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemistry*
  • Luminescent Proteins / chemistry*
  • Metalloproteins / chemistry*
  • Repetitive Sequences, Nucleic Acid
  • Sequence Analysis, DNA*
  • Zinc Fingers

Substances

  • DNA-Binding Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • Metalloproteins
  • DNA