Controlling the fate of synthetic cells

Welcome to the new issue of Plenty of Room! Today, I’m excited to bring you how DNA nanotechnology can be used to control the division of artificial cells!

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Controlling the fate of synthetic cells

One of the ultimate dreams in synthetic biology is the creation of artificial cells, and today’s paper brings us a step closer by combining DNA nanotechnology and droplet systems to create a dynamic, controllable system that mimics some of the core behaviors of real cells.

But let’s go in order. Cells are well organized and dynamic systems, with their organelles, their membranes, and all that. But beyond this, a new type of organization has attracted attention: researchers have discovered cellular droplets assembled by liquid-liquid phase separation (LLPS). In these biomolecular condensates (also called biocondensates), the cellular content self-organizes into membrane-less compartments, that form denser phases compared to the surrounding buffers. It’s a pretty cool concept and something I wasn’t familiar with, and you can read an introduction here!

Now, synthetic LLPS droplets have been used in artificial cells and nanodevices before, but what’s groundbreaking about this paper is how the researchers added temporal control to these droplets, using DNA to mimic the dynamic behavior of living cells. So, how did they do it?

The authors engineered DNA droplets by using Y-shaped branched DNA nanostructures (called them Y_A and Y_B), that self-assemble via their complementary sticky ends. These structures form droplets when they hybridize: a 6-branched DNA linker (LAB) crosslinks the Y_A and Y_B, creating binary-mixed droplets. These mixed droplets can then divide by triggering a cleavage reaction using single-stranded DNA, that starts a strand displacement reaction: in this way, the researchers controlled the division of the DNA droplets!

What’s even more fascinating is how they achieved timing control over the droplet division. Using a time-delay circuit based on RNA inhibition and degradation, they could fine-tune when the droplets divide. The circuit works by using inhibitor RNAs to temporarily stop the division, delaying the cleavage of the DNA linkers (LAB). Over time, RNase H—an enzyme that degrades RNA—breaks down the inhibitor RNAs, freeing up the triggers that allow the droplets to split. The researchers used this mechanism to fine tune the timing of the DNA droplet division, varying the amount of RNase H and the concentrations of the inhibitor RNAs.

But the coolest part for me is the fact that the team was able to control the pathway of the division of the DNA droplets, simply by controlling the timing of it! They created ternary-mixed droplets, this time consisting of three types of Y-shaped DNA nanostructures (adding Y_C into the mix with Y_A and Y_B). At this point, the division can follow two different pathways, depending on which linker is cleaved first:

• Pathway 1: The C-droplet (Y_C) was separated first, followed by the division of the remaining A·B-droplet (Y_A and Y_B).

• Pathway 2: The B-droplet (Y_B) was divided first, followed by the separation of the remaining C·A-droplet (Y_C and Y_A).

This pathway control was achieved by changing the timing of the release of different division triggers, which allowed the researchers to dictate the sequence of droplet division events.

Finally, the authors applied applied these ideas to build a molecular comparator that can compare miRNA concentrations. In this setup, the concentrations of two different miRNAs determined the timing and division pathway:

• If the concentration of miRNA A was higher than miRNA B, Pathway 1 was selected, leading to the division of the C-droplet first.

• Conversely, if miRNA B concentration was higher, Pathway 2 was chosen, with the B-droplet dividing first.

In this way, they could directly compare the concentration of the two miRNA!

This paper has me thinking about the possibility of using similar DNA, RNA or even protein-based network to create artificial cells that mimic living cells, but also about molecular robots that can perform tasks or chemical reactions based on programmable inputs. And of course, targeted drug delivery systems! That’s always a good one.

There is more in the paper, including a lot of simulation results that are also quite informative. So I strongly recommend reading it here!

More room:

• Translating nanotech in vivo: Ever wondered how nucleic acid nanostructures interact with cells in vivo? Wonder no more! This review focuses on their distribution in tissues and cellular responses in rodents, large animals, and humans. It also outlines future research directions to improve efficacy and provides updates on regulatory considerations for clinical applications.

• Fanzy a nuclease? Okay that’s a terrible pun: but this cool study though explores Fanzor, an ωRNA-guided endonuclease with potential for gene editing, found widely in eukaryotes. Researchers describe its structure from three organisms, showing a shared ωRNA interaction interface despite species differences, and a lot more! A lot of promises for gene editing.

• Superworm’s viruses: The pandemic never ended, at least for the Zophobas morio, called superworm. In this study, the authors used cryo-EM to identify a new densovirus as the cause of a disease affecting superworm farms in the U.S. But not all is lost: The research also showed that non-pathogenic strains offer prophylactic protection.

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