DNA Binding by an Intrinsically Disordered Elastin-like Polypeptide for Assembly of Phase Separated Nucleoprotein Coacervates

The formation of condensed phase nucleoprotein assemblies, such as membraneless organelles (MLOs), that contribute to gene regulation and signaling within the cell is garnering widespread attention. A critical technical challenge is understanding how interactions between intrinsically disordered protein (IDP) and nucleic acid molecular components affect liquid-liquid phase separation (LLPS) into nucleoprotein condensates. To better understand the physics of LLPS that drive the formation of biomolecular condensates (known as coacervates), we investigate a model IDP system using a cationic elastin-like polypeptide (ELP), E3, that is engineered to phase separate and bind DNA upon coacervate formation. Using mean field Flory-Huggins (FH) theory, we create ternary phase diagrams to quantify DNA component partitioning within discrete protein- and solvent-rich phases across a range of salt and E3 compositions. We suggest a modified FH theory that combines canonical FH interaction parameters with an approximation of the Debye-Huckel theory to predict the strength of E3-DNA interactions and partitioning with a variable salt concentration. Finally, we establish a simple two-step DNA solution separation/purification assay to highlight the potential utility of our system. This model LLPS biopolymer platform represents an important chemical engineering-based contribution to synthetic biology and DNA technologies, with possible implications for origin of life discussions.

Automated summary

This study investigates the interaction between intrinsically disordered protein and nucleic acid molecular components in driving liquid-liquid phase separation, with a focus on how a cationic elastin-like polypeptide system can bind DNA and form biomolecular condensates. By developing ternary phase diagrams and modifying Flory-Huggins theory, the research predicts the strength of E3-DNA interactions and partitioning under different salt concentrations. Additionally, a simple two-step DNA solution separation and purification assay is established to showcase the potential utility of the system.