GFP-like fluorophores as DNA labels for studying DNA-protein interactions

J Org Chem. 2012 Sep 21;77(18):8287-93. doi: 10.1021/jo301684b. Epub 2012 Sep 11.

Abstract

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • Benzyl Compounds / chemistry*
  • DNA / chemistry*
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Fluorescent Dyes / chemistry*
  • Imidazoles / chemistry*
  • Imidazolines
  • Molecular Sequence Data
  • Nucleotides / chemistry
  • Nucleotides / metabolism*

Substances

  • 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone
  • 3,5-difluoro-4-hydroxybenzylidene imidazolinone
  • Benzyl Compounds
  • DNA-Binding Proteins
  • Fluorescent Dyes
  • Imidazoles
  • Imidazolines
  • Nucleotides
  • DNA
  • Tli polymerase
  • DNA-Directed DNA Polymerase