Duke University researchers developed a modular platform for producing programmable and synthetic biomolecular condensates (non-membrane bound organelles) with tunable material properties for selective cell partitioning. These programmable assemblies were shown to regulate bacterial plasmid expression and inheritance and modulate a protein circuit in mammalian cells. This approach lays the foundation for engineering designer condensates for synthetic biology applications.

The article “Programmable synthetic biomolecular condensates for cellular control” was published in Nature Chemical Biology.

Non-membrane bound

Unlike many organelles, biomolecular condensates, such as P granules and stress granules, are not bound by a membrane. Instead, these condensates can be organized through several different processes. Condensates can selectively add or remove biomolecules (proteins, RNA, and other biopolymers) from colloidal emulsions, gels, liquid crystals, solid crystals, or aggregates inside cells. This makes it possible to control cellular processes in real-time.

In contrast to lock-and-key-type interactions, condensate formation creates a spatially distinct intracellular compartmentalization. This helps keep the condensate contents separate from the cellular environment around them, which is made possible by the presence of a condensate interface. Synthetic condensates can make an environment where only certain functional parts are kept, which controls how cells work. This is shown by the applications of condensate-mediated plasmid partitioning and protein activity modulation.

Synthetic condensates made from naturally occurring intrinsically disordered proteins (IDPs), which are new proteins that don’t form well-defined three-dimensional structures in non-denatured conditions, have been used to control cell growth, reassign codons of certain mRNAs, and control metabolic flow. However, most synthetic condensate research has been done on how they change phases in a test tube, and it has been unclear if they could function as intended in a living cell and be used to program cellular behavior. 

Set phasers

Lead author Yifan Dai and colleagues from the labs of Ashutosh Chilkoti and Lingchong You at Duke University and Rohit Pappu from Washington University in St. Louis present a rational engineering approach for genetically encoded IDPs capable of undergoing responsive phase transitions that can be used for the design of functional condensates in living cells. They showed that IDPs with tunable sequence composition could have their phase behaviors and physical properties designed in a way that makes them useful for specific applications in living cells.

Their modular system comprises three parts: a sticky, resilin-like IDP that can drive phase separation; a specific DNA-binding domain that can recruit a target plasmid; and a dimerization domain that can add more crosslinks to the network to strengthen the material state. 

Plasmid inheritance was applied as a first proof-of-concept example. By dividing the target plasmid in a certain way, the replication machinery was kept out of reach. This caused the plasmid to slowly disappear over time. Importantly, the effectiveness of the process depended on how the condensate was made. More solid-like assemblies led to stronger plasmid partitioning, which led to higher loss rates. Plasmids were rapidly eliminated from the population as more were introduced.

Second, by co-expressing a chimeric fusion of the sticky, resilin-like synIDP and a transcriptional activator, the authors could selectively bring RNA polymerase to the condensates and increase gene expression from the target plasmid. In this situation, more solid-like assemblies showed less expression than fluid ones, including the non-condensate control. This is because they were more rigid and less permeable.

Last but not least, the authors ran some preliminary tests to see if this system could be used with human cells. By selectively attracting a protein that is easy to break down into sticky, resilin-like IDP-based condensates, they could keep the target from breaking down and increase the efficiency of solid-like assemblies.

The authors say that the method is probably generalizable and will help many cell functions that work better when molecules are placed in certain places.

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