Electrostatic Anchoring in RNA-Ligand DesignDissecting the Effects of Positive Charges on Affinity, Selectivity, Binding Kinetics, and Thermodynamics

Targeting RNA with small molecules is an emerging field in medicinal chemistry. However, highly potent ligands are often challenging to achieve. One intuitive strategy to enhance ligand’s potency is the implementation of positively charged moieties to interact with the negatively charged RNA phosphate backbone. We investigated the effect of such “electrostatic anchors” on binding affinity, kinetics, thermodynamics, and selectivity by MST, SPR, and ITC experiments, respectively, with the Ba SAM-VI riboswitch and the Tte preQ1 riboswitch aptamer model systems. RNA-ligand interactions were dominated by enthalpy, and electrostatic anchors had moderate effects on binding affinity driven by faster association rates for higher charged ligands. Despite the observations of loose binding interactions in SPR experiments with multibasic ligands, selectivity over structurally unrelated RNA off-targets was maintained. Therefore, the addition of positively charged moieties is no universal RNA-ligand design principle, but a purposefully implemented ionic RNA-ligand interaction can enhance potency without impairing selectivity.