Nucleobase Clustering Contributes to the Formation and Hollowing of Repeat-Expansion RNA Condensate

RNA molecules with repeat expansion sequences can phase separate into gel-like condensate, which could lead to neurodegenerative diseases. Here, we report that, in the presence of Mg²⁺, RNA molecules containing 20× CAG repeats self-assemble into three morphologically distinct droplets. Using hyperspectral stimulated Raman microscopy, we show that RNA phase separation is accompanied by the clustering of nucleobases while forfeiting the canonical base-paired structure. As the RNA/Mg²⁺ ratio increases, the RNA droplets first expand and then shrink to adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like RNA droplets, the nucleobase-clustered structure is more prominent at the rim, suggesting a continuously hardening process. This mechanism may be implicated in the general aging processes of RNA-containing membrane-less organelles.