GFP-like Fluorophores as DNA Labels for Studying DNA–Protein Interactions

Abstract

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (<b>FBI</b>) and 3,5-bis­(methoxy)-4-hydroxy-benzylideneimidazolinone (<b>MBI</b>) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (<b>dC</b>^<b>MBI</b><b>TP</b> and <b>dC</b>^<b>FBI</b><b>TP</b>) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The <b>MBI</b>/<b>FBI</b>-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 <b>FBI</b> label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent­(exo-) polymerase