Near-infrared fluorescent oligodeoxyribonucleotide reporters for sensing NF-kappaB DNA interactions in vitro

Oligonucleotides. 2008 Sep;18(3):235-43. doi: 10.1089/oli.2008.0135.

Abstract

Two types of reporters for optical sensing of NF-kappaB p50 protein-oligodeoxyribonucleotide (ODN) duplex interactions were designed and compared in vitro. The reporters were based on the effect of fluorescence resonance energy transfer (FRET) between the pair donor Cy5.5 near-infrared (NIR) fluorochrome and either 800CW emitting fluorescence dye acceptor (800CW-Cy), or a nonemitting QSY 21 dye quencher (QSY-Cy). The donor and the acceptor dyes were covalently linked to the complementary oligonucleotides, respectively: Cy dye was conjugated to 3'-thiol, whereas 800CW or QSY21 were conjugated to a hydrophilic internucleoside phosphate amino linker. The reporters were tested initially using recombinant NF-kappaB p50 protein binding assays. Both reporters were binding p50 protein, which protected oligonucleotide duplex from degradation in the presence of exonuclease.The incubation of 800CW-Cy reporter in the presence of control or IL-1beta treated human endothelial cells showed the uptake of the reporter in the cytoplasm and the nucleus. The measurement of NIR fluorescence ratio (i.e. Cy5.5/800CW) showed a partial loss of FRET and the increased Cy5.5 fluorescence in nontreated, control cells. Thus, the specific p50 binding to ODN duplex reporters affected the donor-acceptor fluorochrome pair. NF-kappaB p50 exhibited the protective effect on FRET between NIR fluorochromes linked to the complementary strands of the reporter duplex.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • DNA / metabolism*
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes
  • Genes, Reporter
  • Humans
  • NF-kappa B p50 Subunit / chemical synthesis
  • NF-kappa B p50 Subunit / metabolism*
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism*
  • Recombinant Proteins
  • Umbilical Veins / cytology

Substances

  • Fluorescent Dyes
  • NF-kappa B p50 Subunit
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • DNA