A G-quadruplex–containing RNA activates fluorescence in a GFP-like fluorophore

Spinach is an RNA aptamer analog of GFP that is widely used for fluorescent labeling of cellular RNAs. Crystal structures of Spinach–fluorophore complexes uncover an unusual G-quadruplex RNA fold that is involved in ligand recognition and tuning of Spinach fluorescence properties. Spinach is an in vitro –selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.