Abstract
Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.