TOWARDS AN AUTOMATED METHOD FOR DETERMINING BIOMOLECULAR CONDENSATE PHASE DIAGRAMS

Liquid-liquid phase separation (LLPS) underlies the formation of membrane-less organelles (MLOs), where biological macromolecules are compartmentalized within the cell to regulate biochemical reactions. LLPS can also lead to the self-assembly of several disease-associated proteins on the pathway to the formation of solid aggregates. Therefore, there is great interest in identifying small molecules that modulate biomolecular condensation. However, the systematic evaluation of physical-chemical conditions that interfere with protein solubility and/or molecular crowding, and, therefore, regulate the protein LLPS process is highly laborious. We developed a plate-based automated method for screening for small molecules enabling us to build phase diagrams of recombinant proteins under several physical-chemical conditions. This method has been successfully tested with recombinant human DDX3X, an RNA helicase protein involved in RNA metabolism and associated with neurodevelopmental disorders. We studied the formation of DDX3X biomolecular condensates varying temperature, pH, ionic strength, molecular crowding agents such as PEG and glycerol, and RNA concentration. DDX3X forms condensates at low pH and with PEG 8000, or in the presence of RNA at neutral pH, and 10% glycerol prevents LLPS. As a perspective, this method can also be applied in the identification of LLPS modulators that disrupt aberrant phase transitions while preserving functionally important LLPS. In conclusion, our results will provide a foundation for the rational design of small molecule modulators of protein LLPS with therapeutic value.

This work was supported by the Ministry of Science Technology and Innovation (MCTI) - Brazilian Government.