A time-delay circuit developed by researchers at Science Tokyo enables precise control over the division of synthetic DNA droplets, which mimic biological Liquid-Liquid Phase Separation (LLPS) droplets found in cells. By utilizing a combination of microRNAs (miRNAs) and the enzyme RNase H, the researchers have successfully regulated the timing of droplet division. This breakthrough paves the way for creating artificial cells with advanced functions, such as drug delivery and molecular computing. Many cellular functions in the human body are controlled by biological droplets called Liquid-Liquid Phase Separation (LLPS) droplets. These droplets, made of soft biological materials, exist inside living cells but are not enclosed by membranes like most cell structures. Because they lack membranes, LLPS droplets can adapt quickly to what the cell needs. They can move, divide, and change their structure or contents. Inspired by these unique properties, scientists have developed synthetic LLPS droplets to mimic their biological counterparts.

 

A study published in the journal Nature Communications on August 27, 2024, marks a significant breakthrough in this field. Researchers from Institute of Science Tokyo (Science Tokyo), Japan, developed a method to precisely control the timing of division in synthetic DNA droplets, which mimic biological LLPS droplets. They achieved this by designing a time-delay circuit, where the division of droplets is regulated by a combination of inhibitor RNAs and an enzyme, Ribonuclease H (RNase H). In their approach, the DNA droplets are held together by Y-shaped DNA nanostructures linked via six-branched DNA linkers. These linkers can be cleaved by specific DNA sequences to the linkers used as division trigger DNAs. Initially, the division triggers are bound to single-stranded RNA (ssRNA) molecules called RNA inhibitors. Adding the enzyme RNase H degrades these inhibitors, freeing the division triggers to cut the DNA linkers and initiate droplet division. The researchers successfully achieved pathway-controlled division in a ternary-mixed C·A·B-droplet system, consisting of three Y-shaped DNA nanostructures held together by two linkers. This pathway control was then applied to a molecular computing element known as a comparator, which compared concentrations of microRNA (miRNA) used as inhibitor RNAs.

 

While the study's chemical reactions showed promise, they were temporary and did not sustain a non-equilibrium state like cellular systems. To develop stable and sustainable non-equilibrium systems, researchers emphasize the need for chemical reactions that maintain a continuous supply of energy. Despite this, the research provides a valuable foundation for further advancements in controlling synthetic droplet dynamics.