Phase Separation of RX Repeat Peptides with Nucleic Acids

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Biomolecular liquid-liquid phase separation (LLPS) organizes membraneless cellular compartments.
• Intrinsically disordered proteins, rich in arginine and glycine (RGG/RG), mediate LLPS with nucleic acids, particularly G-quadruplex (G4) DNA.
• The role of glycine in protein-nucleic acid LLPS is less understood than arginine's.

Purpose of the Study:

• To systematically investigate the contribution of glycine residues to LLPS and liquid-solid phase separation (LSPS) with DNA.
• To determine how peptide properties like side-chain size, hydrophobicity, and aromaticity influence phase separation.
• To guide the rational design of peptides for controlled LLPS with nucleic acids.

Main Methods:

• Systematic substitution of glycine residues with alanine, proline, valine, and tyrosine in RX repeat peptides.
• Turbidity and microscopy assays to observe phase separation.
• Testing interactions with DNA oligonucleotides forming G4, duplex, and random coil structures.

Main Results:

• RP and RA peptides enhanced LLPS with G4 DNA compared to RG peptides.
• RY peptide promoted liquid-solid phase separation (LSPS) with G4 DNA but LLPS with random coil and duplex DNA.
• RV peptide formed aggregates independently of DNA, while alanine and proline substitutions modulated LLPS/LSPS selectivity.

Conclusions:

• Peptide side-chain characteristics (size, hydrophobicity, aromaticity) are critical for selective LLPS and LSPS with DNA secondary structures.
• Understanding these factors provides mechanistic insights into protein-nucleic acid interactions.
• This knowledge aids in designing peptides for specific phase separation behaviors, distinguishing between LLPS and LSPS.