Phase Separation of RX Repeat Peptides with Nucleic Acids

Biomolecular liquid-liquid phase separation (LLPS) plays a crucial role in organizing membraneless cellular compartments, which regulate a wide variety of cellular processes. A key molecular mechanism underlying LLPS of nucleic acids involves G-quadruplex (G4) structures of DNA and RNA interacting with intrinsically disordered proteins, particularly arginine and glycine (RGG/RG) rich proteins. The role of arginine residues in LLPS has been studied extensively, whereas few studies have focused on the role of the another frequently occurring residues, glycine. Here, we systematically investigated the contribution of G residues by substituting them with alanine (A), proline (P), valine (V), and tyrosine (Y) residues, generating a series of RX repeat peptides. Turbidity and microscopy assays with DNA oligonucleotides forming G4, duplex, as well as random coil, showed that RP and RA-peptides enhanced LLPS with G4 DNA, by comparing RG-peptide. In contrast, RY promoted liquid-solid phase separation (LSPS) with the G4 DNA, although it underwent LLPS with the random coil and duplex DNAs. In addition, RV-peptide formed aggregates even in the absence of any DNA. These results demonstrate that side-chain size, hydrophobicity, and aromaticity are critical factors for the LLPS and LSPS capability and selectivity with DNA forming various secondary structures. This study provides mechanistic insights into protein-nucleic acid LLPS and LSPS and guides the rational design peptides to undergo LLPS but not LSPS with nucleic acids.