A piece of RNA dynamically folds into a secondary structure determined by its sequence. Interactions between nucleotides mold stretches of RNA into hairpin loops, triplexes, and switches, among other forms. The predictability of the folding and the range of geometries possible with different folds are not unlike the art of origami. This idea underlies RNA origami, a technique for folding RNA into custom nanostructures.

“You fold the RNA scaffold with short RNA or DNA staple strands into a design target shape,” said Emanuela Torelli, a synthetic biologist at Newcastle University. “The principle is the same as DNA origami, but there are a few things that you may need to consider because the helix has different features.”

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DNA origami involves folding single-stranded DNA and is a better-established field. However, chemical and structural differences between DNA and RNA mean that applying lessons from DNA origami to RNA molecules opens up new nanostructure geometries and applications.

Folding RNA into Diverse Structures

Compared to DNA, RNA is far less stable. Those who have handled RNA in labs know how incredibly difficult it is to keep it intact. A major reason for its high instability is the 2' hydroxyl group on the RNA molecule. This is why the RNA origami scaffold often needs to be stabilized with DNA staples in what is sometimes called DNA-RNA hybrid origami.

Another difference is the twist on the helices. “DNA usually adopts B-form conformation, RNA is usually in A-form,” said Anna Romanov, a graduate student at MIT. In DNA, the base pairs are concentrated over the helical axis, whereas they are spaced away in RNA. “If you're making DNA-RNA hybrid origami, you have to think about the B-form versus the A-form design,” added Romanov.

The high reactivity of RNA is also a strength when it comes to RNA origami. “Due to the hydroxyl group, RNA folds back upon itself in more interesting ways than DNA can,” said Thom LaBean, CSO at North Carolina-based startup Helixomer.

The reactivity also makes RNA origami modular. Smaller RNA structures can be combined to create more complicated structures. Some intricate shapes that researchers have fashioned by folding RNA include honeycombs, nanofibers, and five-sided double pyramids. These nanostructures can be functionalized by adding chemical entities on RNA origami scaffolds. While this kind of bottom-up synthetic biology is viable with DNA origami, RNA offers a greater range of interactions as it is more chemically versatile and catalytic.

Most DNA origami designs are multi-molecular, with different DNA sequences stitched together with staple strands, and therefore don't make for good drugs. “You don't want to put a supramolecular complex of DNA into the bloodstream because it's going to come apart a lot easier,” said LaBean. RNA origami structures, on the contrary, can be folded from a single molecule.

Inside cells, many RNAs fold co-transcriptionally. In other words, they assume their structure as they are being synthesized. The same logic can be applied to RNA origami, thereby making it programmable.

It begins by routing a single RNA strand to approximate a desired shape. “Then you assign a sequence to that strand such that it folds correctly and translate the sequence into a DNA template that can be transcribed,” explained Kerstin Göpfrich, a biophysicist at the University of Heidelberg. Not only is co-transcriptional RNA origami compatible with existing genetic engineering tools, but it allows the bioproduction of larger structures. 

RNA origami nanostructures can be made to mimic cellular structures. For example, Göpfrich’s research group is trying to build molecular hardware for synthetic cells with RNA origami. “Those structures could serve as genetically encodable biophysical tools for manipulating living cells,” added Göpfrich.

But folding an RNA is only part of the puzzle. The folded mRNA that encodes a particular gene needs to be self-assembled to have it deliver itself and then express the protein of interest. Currently, “that's a bit harder because you have to account for how the new structure of the molecule affects downstream translation,” said Romanov.

Applications in Therapeutics and Gene Editing

RNA origami opens up interactions relevant to drug-target interactions that are not usually achievable with DNA origami. These include delivery systems with controlled release kinetics or triggers for immune cells. “With growing interest in RNA-based medicines, I see great prospects in RNA origami-based vaccines or drugs in the emerging field of synthetic immunology,” said Göpfrich.

While the immunogenicity of RNA molecules is beneficial when developing vaccines, it can be a challenge for other therapeutic applications. “If you’re trying to deliver other drugs or genes, then having this inflammation is generally a bad thing, and you want to be able to minimize the innate immune stimulation,” said Romanov.

Helixomer is developing an RNA origami drug that addresses this challenge by incorporating 2’ fluoropyrimidines into the origami structure. This modification makes it less likely for the RNA to elicit an immune response and increases its stability. Anticoagulants or blood thinners act by binding and inhibiting the activity of thrombin, an enzyme that encourages the formation of blood clots. Helixomer places thrombin-binding aptamers on an RNA origami structure.

“The cool thing about the origami is that we can control the orientation and the spacing of the aptamers to get higher affinity,” said LaBean. A side effect of anticoagulants is excessive bleeding, necessitating reversing the drug mechanism. The other part of the drug combination is a DNA molecule that binds the aptamers on the RNA scaffold and quickly reverses the anticoagulation activity.

Researchers are leveraging RNA origami to deliver mRNA sequences or silence genes. “You can assemble mRNA into a specific shape and eventually deliver this nanostructure to a specific cell line,” said Torelli. “Or you can add specific sequences, like silencing RNA sequences, to ​an RNA origami structure.”

The technology can also facilitate controllable gene expression. For example, in a study published in the journal Nucleic Acids Research, Aarhus University researchers attached single guide RNAs for CRISPR-editing genes across different metabolic pathways on a single RNA origami scaffold. Changing the orientation of the scaffold altered gene expression by impacting the amount of transcription factors that bound to it.

Lastly, RNA origami is advancing our understanding of RNA biology. Notably, researchers are probing how manipulating individual bases changes RNA origami nanostructures. These insights, in turn, could improve the design of RNA origami and other DNA and RNA-based nanostructures.

What’s Next for RNA Origami?

The COVID pandemic unwittingly accelerated RNA origami. Firstly, the success of mRNA vaccines renewed interest in RNA-based medicines. Secondly, it led to pharma companies setting up production lines for the enzymatic synthesis of RNA strands, a component crucial for RNA nanotechnology.

Moreover, advances in co-transcriptional RNA origami simplify the scaling up of RNA origami production. When it comes to building with biomolecules, the advantage of RNA (or DNA) origami over proteins is that nucleic acids have fewer characters and, consequently, more straightforward folding rules. Over the last few years, advances in AI-based protein design tools have narrowed that gap. However, AI is also coming for RNA sequences and could be used to optimize the design of scaffold and staple sequences.

“The next generation of experiments would be focused on combining  AI and RNA origami design to find novel therapeutic molecules,” said Torelli. What we learn could advance bottom-up nanofabrication for applications extending beyond medicine.