Site-Specific Turn-On Fluorescent Labeling of DNA-Interacting Protein Using Oligodeoxynucleotides That Modify Lysines To Produce 5,6-Dimethoxy 3-Methyleneisoindolin-1-one.

Abstract

We have developed oligodeoxynucleotides (ODNs) that modify primary amines to produce 5,6-dimethoxy 3-methyleneisoindolin-1-one. Compared to the oxygen isosteric fluorophore, 4,5-dimethoxyphthalimide, this methyleneisoindolinone was more stable and exhibited an 85 nm blue-shifted fluorescent emission (λmax at 425 nm) with an intensity comparable to that of the phthalimide. Reaction of the DNA-binding domain of Escherichia coli DnaA protein with an ODN containing its binding sequence efficiently afforded a modified fluorescent protein at a specific lysine residue in the proximity of the ODN. A full-length DnaA protein was also successfully fluorescently labeled. These results demonstrate the potential utility of the ODNs developed in this study for the fluorescent labeling of DNA-interacting protein at the lysine residue of interest.