A Tandem Green-Red Heterodimeric Fluorescent Protein with High FRET Efficiency

Abstract

The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato-a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green-red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99% intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live-cell imaging applications.