Probing the molecular determinants of emission enhancement in RNA aptamer-metal complex systems.

Abstract

Aptamer recognition of metal complexes is distinct from organic molecules, as it requires the nucleic acid to provide complementary interactions based on both the metal-centered geometry and the steric and electronic features of the ligands. Molecular determinants for aptamer recognition, specificity, and productive binding of coordination complexes are poorly understood. Here, we examined the promiscuity and discrimination of the 12Rd5 aptamer, evolved to bind [Ru(bpy)₃]²⁺ (bpy = 2,2'-bipyridine), using structurally related metal complexes. Emission enhancement, equilibrium dialysis, and isothermal titration calorimetry revealed that 12Rd5 accommodates changes in the metal center and net charge on the complex but strongly discriminated between different ligands. Compounds 1-3 exhibited emission enhancement and formed long-lived interactions with the aptamer, with half-lives exceeding 20 h, while analogs with expanded π-surfaces or additional heteroatoms in the ligands failed to engage the aptamer productively. Mutagenesis and truncation of the RNA revealed that even minimal perturbations abolish binding, indicating that recognition is governed by a highly cooperative global fold rather than local sequence motifs. AlphaFold 3 modeling and docking provided limited structural insight, highlighting persistent challenges in RNA structure prediction and a need for additional training data for predictions involving metal complexes. These results demonstrate that a single evolved aptamer can accommodate multiple coordination compounds within specific geometric and electrostatic constraints, providing foundational information for the repurposing or redesign of hybrid inorganic-aptamer-based sensors, imaging agents, and metal-mediated biochemical tools.