Structure of a dopamine-binding RNA aptamer reveals metal-mediated ligand recognition

Abstract The selection of small-molecule binding RNA aptamers enables the creation of ligand-responsive RNA tools, yet most aptamers fail to operate reliably across diverse environments. Scaffolded selection addresses this limitation by preserving the tertiary architecture of a riboswitch scaffold while driving the evolution of a new ligand-binding pocket. Using the xpt purine riboswitch aptamer, we previously generated dopamine-binding aptamers. Here, we report the crystal structures of two representative variants in their apo and dopamine-bound states to define how they recognize ligand while maintaining scaffold integrity. The structures demonstrate that scaffolded selection enforces global fold conservation and retains the defining tertiary interactions of the parental riboswitch. Local remodeling, triggered by deletions acquired during selection, rewires the three-way junction to build extensive interaction networks that host the dopamine-binding pocket. A deeply buried potassium ion anchors ligand recognition by coordinating the dopamine hydroxyl group while the RNA engages the catechol ring through stacking and hydrogen bonding interactions. Structure probing shows minimal conformational changes upon ligand binding, indicating that the aptamers adopt a largely preorganized fold. These findings strengthen the central premise of scaffolded selection: riboswitch-derived “superfolder” architectures can bias in vitro selections towards aptamers that conserve global structure while supporting locally diverse binding pockets. This balance between structural stability and local plasticity expands the capacity of a single RNA fold to recognize chemically distinct ligands and positions scaffolded selection as a powerful platform for engineering robust RNA-based sensing and regulatory devices. TOC Abstract (graphical)