Fast, Bright, and Reversible Fluorescent Labeling of Rhodamine-Binding Proteins

High Resolution Image Download MS PowerPoint Slide Rhodamine dyes conjugated to targeting ligands can yield exceptionally bright fluorescent probes for live-cell imaging. However, the limited permeability of such rhodamine derivatives restricts their broader applications, particularly in vivo . Here, we present Rho-tag and SiR-tag, engineered protein tags derived from bacterial multidrug-resistant proteins that bind unsubstituted (silicon) rhodamines with nanomolar affinity. Unsubstituted (silicon) rhodamines readily cross membranes and enable rapid, reversible, and fluorogenic labeling of the tags in mammalian cells within seconds. The labeling of Rho-tag and SiR-tag is compatible with various super-resolution imaging methods and allows their use alongside self-labeling tags, such as HaloTag7 and SNAP-tag. The high affinity and specificity of both tags, combined with the permeability and outstanding spectroscopic properties of rhodamines, make them particularly attractive for in vivo bioimaging, as demonstrated by efficient fluorescent labeling in C. elegans embryos and zebrafish larvae.