Water-detected NMR allows dynamic observations of repeat-expansion RNA condensates

RNA condensation is implicated in the formation of neurotoxic RNA foci in cells affected by genomic expansions of trinucleotide or hexanucleotide repeats. However, the biophysical properties of repeat-expansion RNA condensates are poorly understood. Using CAG repeat-expansion RNA as a model system, we show that these RNA condensates cannot be observed with conventional nuclear magnetic resonance techniques. Therefore, we developed a nuclear magnetic resonance approach, based on water-detected semi-solid magnetization transfer, to detect and characterize RNA condensates in vitro. Our method, termed condensate detection by semi-solid magnetization transfer (CONDENSE-MT), is broadly applicable, highly sensitive and does not require direct observation of the biomolecules of interest. Using CONDENSE-MT, we could obtain dynamic information about RNA condensates, such as the relative amount and the tumbling rate of condensed RNA, the proton-solvent exchange kinetics and the amount of water molecules transiently bound in the condensate. We find that phase separation dramatically decreases molecular tumbling and is driven by heterotypic interactions between RNA and Mg²⁺. We further show that increasing CAG repeats decreases condensate hydration.