RNA Skeletons & Artificial Cells: Pushing Synthetic Biology's Limits

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Today we dive deep into synthetic biology! What if I told you we can make artificial cells with RNA skeletons?

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RNA skeletons & artificial cells

I said a while ago that I don’t cover nearly enough RNA science: compared to proteins and DNA, I have not given this building block of cells nearly enough love. And today I’m changing that! But let’s start from the beginning.

Building cells from scratch: the synthetic biology challenge

One of synthetic biology’s most ambitious goals is to create an artificial cell from non-living molecular components. As you might imagine, this is hard! But the possible advantages are worth it: such a system would offer unprecedented control over biological systems, and it would revolutionize medicine, biotechnology and more.

A key challenge is building stable cellular structures inside artificial cells. Natural cells rely on protein cytoskeletons to maintain their shape and mechanical stability, to facilitated intracellular transport and organize biochemical reactions. Unfortunately, copying this blueprint in synthetic cells is not easy (like always in biology). For example, the protein production machinery alone requires over 150 genes! Definitely not a viable minimal system.

Can we avoid proteins?

One solution to this problem is to remove proteins completely: scientists have tried using DNA origami to build cellular scaffolds. But DNA has its own limitations:

• DNA doesn’t have a lot of functionalities, unless is chemically modified.

• Using DNA both as a genetic and functional material makes design complicated.

• It’s impossible to implement evolution, a distinct (and cool) feature of living systems.

As you might have noticed, this leaves us one last building block: RNA. Finally, the solution!

RNA origami as a cytoskeleton

This must be what the researchers behind today’s paper thought. Here, they created RNA cytoskeletons for artificial cells using RNA origami. I cover a lot of DNA origami in this place, but RNA origami is just as cool, and in some ways better. Unlike DNA origami, RNA origami folds as it is transcribed, meaning the RNA strand naturally adopts its designed shape without external assistance. Super cool!

RNA is a good alternative to both DNA and proteins because:

• RNA origami folds co-transcriptionally, eliminating the need for complex folding systems, like chaperones for proteins or heat for DNA origami.

• RNA structures are encoded in DNA and can be produced inside synthetic cells.

• RNA creates predictable 2D and 3D structures: ideal for programmable nanostructures!

So, where did the team start from?

Designing RNA nanotubes and unexpected discoveries

The researchers engineered RNA nanotubes, using repeating RNA tiles that self-assemble into rigid, long and thin filaments: nanoscale reinforcement rods! The sequences of the RNA were optimized so that the nanotubes are formed as soon as the RNA is synthetized. The RNA tiles contained kissing-loop interactions, a common RNA structural motif that stabilizes complex architectures and helped the tiles to lock together into long chains. Using this technique, the researchers created different versions of nanotubes, with length ranging from 700 nm to almost 1 micrometer! It’s crazy to think they are creating them using tiles that are only 11 nm in length! There must be a lot of them. They used atomic force microscopy and molecular dynamics simulations to confirm that the structures assembled as designed: and while at it, they also simulated the largest RNA structure to date!

Funnily enough, while optimizing the RNA nanotubes, the researchers noticed that some RNA variants assembled into closed RNA nanorings instead of linear nanotubes. Like true scientists, they didn’t miss the opportunity to study something unexpected: they figured out that the RNA sequences that formed ring contained weaker bonds. This meant that it was energetically more convenient to form closed rings, rather than elongating into long filaments. The nanorings could be a new architecture for use in artificial cells!

RNA cytoskeletons: finally inside (artificial) cells!

Going back to the nanotubes, the researchers moved on to mimic a self-sustaining, controllable synthetic cell. To do this, they combined the RNA nanotubes with lipid vesicles (giant unilamellar vesicles, GUVs). The team encapsulated DNA templates inside the GUVs. Then, by adding magnesium salts, they triggered RNA transcription inside the vesicles, causing RNA nanotubes to self-assemble.

The result? A beautiful, fluorescent network inside the vesicles: over the course of a few hours, the cells built their own cytoskeleton from RNA!

The team wanted to added a final functional twist to their system. Inspired by the natural cell cortex, which interacts with the cell membrane and shapes the cell, they attached a protein to the nanotubes so they could bind to the vesicles. In this way, the nanotubes could exert mechanical force on the vesicles membrane and changed its shape, much like protein cytoskeletons in natural cells!

Engineering dynamic synthetic cells: future directions

This cool study is a big step forward in synthetic biology, demonstrating that RNA origami can serve as an intracellular cytoskeleton in artificial cells. RNA is an interesting material because it can be expressed directly inside cells, and this expression can be controlled easily. In addition, it could also work as a scaffold for enzymatic reactions and metabolic pathways. This could lead to applications in a few fields:

• Drug delivery: RNA-based vesicles could be engineered to release cargo in response to environmental cues.

• Molecular sensing: RNA nanostructures could be designed to detect and respond to specific biomolecules.

• Biophysics research: RNA cytoskeletons provide a simpler platform to study cytoskeletal mechanics without the complexity of proteins.

It was an interesting work! And I feel like I couldn’t actually make it justice, so go and read it here!

And as always, thank you for reading! What are your thoughts on RNA origami? Are you a DNA origami loyalist? Did you enjoy this paper? Reply and let me know!

More room:

• From balls to stars and back: This paper has a special place in my heart: they used the software I developed during my PhD! Good thing it’s still working. The team created a wireframe DNA origami that reversibly transforms between open and closed forms using DNA hybridization and strand displacement. Spacer strands introduce flexibility, enabling controlled shape changes. Simulations and microscopy confirmed successful assembly and transformation. This advance in dynamic DNA nanostructures could lead to molecular devices and nanoscale robotics.

• Hexameric helicase unveiled: I love structural biology! And this study uses cryo-EM to reveal how the LTag helicase initiates DNA unwinding at replication origins. LTag forms head-to-head hexamers, melting DNA at two sites to create bidirectional forks. The helicase pulls one DNA strand through its channel while expelling the other, driven by ATP hydrolysis as an entropy switch rather than direct mechanical force. These findings provide a model for replication fork progression and uncover key principles of ATP-dependent molecular machines.

• DNA origami for bio and drugs: If you are unsure what to do with DNA origami, this is the review for you (and for me, since I’m also reading it). It explores current methods for functionalizing, purifying, and characterizing DNA origami nanostructures, highlighting challenges such as limited functionalization efficiency and characterization difficulties. The discussion emphasizes areas where further research can enhance the fabrication and application of functionalized DNA origami.

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