Spectral tuning by a single nucleotide controls the fluorescence properties of a fluorogenic aptamer.

Fluorogenic aptamers are genetically encoded RNA aptamers that bind and induce the fluorescence of otherwise nonfluorescent small molecule dyes. These RNA-fluorophore complexes can be highly fluorescent and useful for RNA visualization and genetically encoded biosensors. Notably, different RNA aptamers can bind the same fluorophore resulting in complexes that exhibit spectrally distinct fluorescence properties. The basis for spectral tuning of small molecule fluorophores has not yet been studied. Here we explore the mechanism of spectral tuning in three highly related RNA aptamers, Broccoli, Orange Broccoli, and Red Broccoli, each of which binds the DFHO (3,5-difluoro-4-hydroxybenzylidene imidazolinone-2-oxime) fluorophore, and generates distinct spectral emissions. Here we show that DFHO fluorescence spectral tuning is controlled by interaction of the oxime moiety of the fluorophore and one specific nucleotide that is different in each RNA aptamer. Our finding presents, for the first time, a mechanism by which RNA can control the properties of a bound small molecule fluorophore. More broadly, our finding can guide further development of fluorogenic aptamers with novel spectral properties.