Peptide-directed folding of the elusive RNA i-motif.

Folded RNA structures are increasingly being recognised as key regulators in biological processes, yet the RNA i-motif remains poorly characterised due to its low stability and lack of selective molecular probes. Here, we describe the first ligand - a short peptide - that binds the elusive RNA i-motif. Our minimalist peptide RGGFGGRGG is derived from the intrinsically disordered region of the protein nucleolin and binds to folded RNA over DNA with >5-fold selectivity. The binding of two peptide molecules folds the RNA i-motif at a higher pH than under native conditions. This folded, peptide-bound structure can still bind other guests, such as the intercalator thiazole orange, displaying heteroallosteric properties. Our peptide binding is driven by more than simple electrostatic attraction, exploiting the subtle differences in steric complementarity and hydration of the compact RNA structures relative to DNA congeners and unfolded strands. Our findings underline the potential of minimalistic peptide scaffolds as selective binders for non-canonical RNA structures, allowing for the probing and modulation of RNA topologies.