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.

甜菜根是一种同二聚体外选择的 RNA，可结合并激活 DFAME（一种源自 GFP 的条件荧光团）。它与之前鉴定的同源二聚适体 Corn 有 70% 的序列相同性，Corn 在其原体间界面结合了同源荧光团 DFHO 的一个分子。我们现在已经以 1.95 Å 的分辨率确定了甜菜根-DFAME 共晶结构，发现这种 RNA 同二聚体在相距约 30 Å 的位点结合荧光团的两个分子。除了这种整体结构差异之外，甜菜根和玉米的非规范、复杂四链体核心的局部结构也明显不同，这强调了微妙的 RNA 序列差异如何导致意想不到的结构差异。通过结构引导工程，我们生成了一种对 DFHO 具有 12 倍荧光激活选择性开关的变体。甜菜根和这种变体形成异二聚体，并构成工程标签的起点，其贯穿空间的荧光团间相互作用可用于监测 RNA 二聚化。