Red‐Shifted Substrates for FAST Fluorogen‐Activating Protein Based on the GFP‐Like Chromophores

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen‐activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)‐like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580–650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes. Red on arrival: A genetically encoded fluorescent tag with red signal capable of live cell microscopy is presented. The tag is based on previously published fluorogen‐activating protein FAST and a novel fluorogenic derivative of GFP‐like chromophore. The proposed dye instantly stains proteins fused with FAST, washes out in a minute timescale, and exhibits high photostability of the fluorescence signal.