A Multicolor Large Stokes Shift Fluorogen‐Activating RNA Aptamer with Cationic Chromophores

Abstract Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4‐hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes‐shifted emission from several new green and red HBI analogues that are non‐fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO + , which binds to the Chili aptamer with a low‐nanomolar K D . Because of its highly red‐shifted fluorescence emission at 592 nm, the Chili–DMHBO + complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET‐based analytical RNA systems.