title: GFP-like fluorophores as DNA labels for studying DNA-protein interactions.
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.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Riedl
authors.forename: Petra
authors.initials: P
authors.lastname: Ménová
authors.forename: Radek
authors.initials: R
authors.lastname: Pohl
authors.forename: Petr
authors.initials: P
authors.lastname: Orság
authors.forename: Miroslav
authors.initials: M
authors.lastname: Fojta
authors.forename: Michal
authors.initials: M
authors.lastname: Hocek
mesh_terms: D020029:Base Pairing; D001593:Benzyl Compounds / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D048288:Imidazolines; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*
chemical_list: C579736:3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone; C560120:3,5-difluoro-4-hydroxybenzylidene imidazolinone; D001593:Benzyl Compounds; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007093:Imidazoles; D048288:Imidazolines; D009711:Nucleotides; D004247:DNA; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase