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, and this process may lead to neurodegenerative diseases. Here we report that in the presence of Mg2+ ion, RNA molecules containing 20CAG repeats coacervate into filled droplets or hollowed condensate. Using hyperspectral stimulated Raman spectroscopy, we show that RNA coacervation is accompanied by the stacking and clustering of nucleobases, while forfeiting the canonical base-paired structure. At an increasing RNA/Mg2+ ratio, the RNA droplets first expand in sizes, and then shrink and adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like droplets, the nucleobase-clustered structure is more prominent at the rim than at the center, accounting for the rigidification of RNA droplets. Thus, our finding has broad implications for the general aging processes of RNA-containing membrane-less organelles.