Fig. 1: Concept of the regenerative and fluorogenic array tag ReTagX.

In conventional self-labeling tags, fluorescent probes are covalently linked to the protein tag, leading to irreversible photobleaching under intense illumination. Array tags, which recruit multiple reporters, can exhibit a tenfold increase in brightness and extended tracking duration, but their large size (~1.4 mDa) may hinder protein diffusion and interactions. ReTagX (18–156 kDa) developed in this study consists of multiple repeats of a regenerative protein tag paired with highly fluorogenic rhodamines. These rhodamines exist in a non-fluorescent spirocyclic form when unbound, significantly reducing background signals and creating a pool of fresh probes. Upon binding to ReTagX, the rhodamines transition to their fluorescent zwitterionic states. Through reversible labeling with ReTag1, bleached dyes rapidly dissociate from the tag, allowing fresh probes to bind and enabling fluorescence self-recovery. In array tag ReTagX, even when one fluorophore was bleached or exchanged, other fluorophores on ReTagX continued to fluoresce, significantly enabling extended imaging duration. Created with BioRender.com.