using-solution-nmr-to-characterize-biomolecular-condensates-under

Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions

Biomolecular condensates formed through liquid&#8211;liquid phase separation (LLPS) organize the intracellular environment and regulate diverse ...

ETH Zurich

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 Mg. We further show that increasing CAG repeats decreases condensate hydration.

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

Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components, such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labeling of various droplet components, which may affect phase separation behavior and are thus not able to probe biomolecules in their chemically native state. Here, we introduce a noninvasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both the dilute and condensed phases. We demonstrate the robust performance of this 1D H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.

Article	Year
Water-detected NMR allows dynamic observations of repeat-expansion RNA condensates. Nature chemistry\| PubMed ID: 41094266	2025
Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag. Journal of the American Chemical Society\| PubMed ID: 41360394	2025

Johannes Schmoll

Biography

JoVE Journal Publications

Other Publications