A time-delay circuit has been created that allows for accurate management of how synthetic DNA droplets divide, which serve as models for the biological Liquid-Liquid Phase Separation (LLPS) droplets present in living cells. Using a mix of microRNAs (miRNAs) and the enzyme RNase H, researchers have effectively controlled the timing of this droplet division. This innovation opens up new possibilities for developing artificial cells with enhanced capabilities, like drug delivery and molecular computing.
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.
In the human body, various cellular functions are managed by biological droplets known as Liquid-Liquid Phase Separation (LLPS) droplets. These droplets consist of flexible biological materials that exist in living cells but are not surrounded by membranes like typical cellular structures. Because they lack membranes, LLPS droplets can quickly adapt to the cell’s requirements, moving, dividing, and altering their structure or contents. This adaptability is crucial for numerous functions, including the transcription of ribosomal RNA (rRNA) in the nucleolus, facilitating sol-gel transitions where materials change between fluid and gel states, and managing chemical reactions within cells.
Motivated by these remarkable characteristics, researchers have created synthetic LLPS droplets to replicate their biological relatives. Although there has been notable advancement in controlling synthetic droplet movement and division, achieving precise timing in these processes has been a challenge.
A study released in the journal Nature Communications on August 27, 2024, represents a significant advancement in this area. Researchers from the Institute of Science Tokyo (Science Tokyo), Japan, have formulated a method to accurately control the timing of synthetic DNA droplet division, taking cues from biological LLPS droplets. They created a time-delay circuit that regulates droplet division using a mix of inhibitor RNAs and the enzyme Ribonuclease H (RNase H).
Professor Masahiro Takinoue, the study’s lead author, explains: “We demonstrate the timing-controlled division dynamics of DNA droplet-based artificial cells by coupling them with chemical reactions exhibiting a transient non-equilibrium relaxation process, resulting in the pathway control of artificial cell division.”
In their method, the DNA droplets are held together by Y-shaped DNA nanostructures connected through six-branched DNA linkers. Specific DNA sequences serve as division triggers that can cleave these linkers. Initially, the division triggers are combined with single-stranded RNA (ssRNA) called RNA inhibitors. When the enzyme RNase H is added, it degrades these inhibitors, releasing the division triggers to cut the DNA linkers and start droplet division. “These two reactions create a time delay in the cleavage of the DNA linker, resulting in precise control of DNA droplet division,” elucidates Takinoue.
The researchers successfully obtained pathway-controlled division in a ternary-mixed C·A·B-droplet system, comprising three Y-shaped DNA nanostructures connected via two linkers. By manipulating the inhibition and release of division triggers, they developed two specific division pathways: Pathway 1, where C·A·B-droplets first split into C-droplets followed by A·B-droplets, and Pathway 2, where C·A·B-droplets initially split into B-droplets and then C·A-droplets.
This pathway control was applied to a molecular computing element known as a comparator, which assesses the concentrations of microRNA (miRNA) acting as inhibitor RNAs. The comparator used variations in RNA concentrations to determine the division pathway taken, offering a way to quantitatively compare RNA levels, with prospective uses in diagnostics.
While the chemical reactions in this study showed promise, they were temporary and did not persist in a non-equilibrium state like those found in cellular systems. To develop systems that are stable and sustainable in non-equilibrium states, researchers highlight the necessity for chemical reactions that continuously provide energy. Nonetheless, this study lays a critical groundwork for further developments in managing synthetic droplet dynamics.
“We believe that this technology provides a strategy to create artificial cells and molecular robots with more sophisticated functions, such as timing-controlled self-replication, drug delivery, and diagnosis, with more accuracy and quantitative specifications,” states Takinoue.
