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

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.

Förster resonance energy transfer; RNA aptamers; fluorescence; fluorescent proteins; large Stokes shift.