Discrimination between Purine and Pyrimidine-Rich RNA in Liquid–Liquid Phase-Separated Condensates with Cationic Peptides and the Effect of Artificial Crowding Agents

Membraneless organelles, often referred to as condensates or coacervates, are liquid-liquid phase-separated systems formed between noncoding RNAs and intrinsically disordered proteins. While the importance of different amino acid residues in short peptide-based condensates has been investigated, the role of the individual nucleobases or the type of heterocyclic structures, the purine vs pyrimidine nucleobases, is less researched. The cell's crowded environment has been mimicked <i>in vitro</i> to demonstrate its ability to induce the formation of condensates, but more research in this area is required, especially with respect to RNA-facilitated phase separation and the properties of the crowding agent, poly(ethylene glycol) (PEG). Herein, we have shown that the nucleotide base sequence of RNA can greatly influence its propensity to undergo phase separation with cationic peptides, with the purine-only RNA decamer <b>(AG)</b>_<b>5</b> readily doing so while the pyrimidine-only <b>(CU)</b>_<b>5</b> does not. Furthermore, we show that the presence and size of a PEG macromolecular crowder affects both the ability to phase separate and the stability of coacervates formed, possibly due to co-condensation of PEG with the RNA and peptides. This work sheds light on the presence of low-complexity long purine- or pyrimidine-rich noncomplementary repeat (AG or CU) sequences in various noncoding RNAs found in biology.