Supersize DNA origami

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It’s time we go back to DNA origami, but what if we supersize them? Today we check out DNA origami superstructures!

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Supersize DNA origami

After some exciting AI-driven enzyme design, it’s time to go back to some good old DNA origami! I, for one, am excited.

DNA nanotech is probably the best method to precisely design nanoscale structures from the ground up, and DNA origami is for sure the most used technique. By folding a long single-stranded DNA (ssDNA) scaffold is folded into shape using short staple strands, researchers have used DNA origami nanostructures for just about everything: from cancer drugs and vaccines to reshaping microvesicles, and more. However, one of the fundamental limitations of the technique is size:

• The maximum size of a single structure is constrained by the scaffold length: these are generally based on the M13 phage and they are between ~7000 and ~9000 bases, with some common scaffolds pushing ~10000.

• It’s hard to maintain full addressability (precise molecular positioning) while increasing structure size.

• Several methods are available to scale up DNA origami structures, each with pros and cons:

  • Hybrid scaffold (Lambda/M13 DNA): these increase the scaffold length up to 50000 base pairs! But they also require a much more complex synthesis and the number of staples become very high, making it more expensive.

  • Hierarchical assembly: One can combine multiple origami structures, creating huge and complex structures, but since there are repetitive units, the full addressability is lost, and often the yield is low, given the multiple steps required.

  • Single-stranded tile assembly: this method enables the controlled creation of the largest structures to date, but it is extremely costly (over $100 000! You get an enormous structure, but still). Plus, this approach is only limited to 2D structures.

In synthesis, there is a need for a scalable, cost-effective and highly controlled method to create large, full addressable DNA nanostructures. And this is why the authors of today’s paper introduce the modular DNA Origami (moDON) approach. Their aim is to overcome the current limitations by combining 3 directional modularity, cost-effective design and one step assembly! Let’s take a closer look at their approach.

The moDON approach is based on a single, cylindrical DNA origami structure, which design can be divided in 3 parts:

• Central core: Acts as the structural backbone and it constitutes around 98% of the structure. This part is identical in all monomers, reducing costs by reusing staple strands.

• Modular shell: This forms the outer layer and it contains programmable connection sites for the lateral assembly. It’s build from modular indentations and protrusions, allowing monomers to interlock in the xy-plane like puzzle pieces. This layer can be easily reconfigured, enabling tens of thousands of unique variants.

• Connection interface: Lastly, each monomer has 3 ssDNA handles which can hybridize with each other and allow stacking in the z-direction.

A key innovation is that the xy- and z-connection systems are completely orthogonal, meaning they don’t interfere with each other. This allows for unprecedented versatility, with nearly 60 000 unique monomer configurations from a single staple set with minimal changes!

The authors first tested the 2 connections systems separately, by creating different structures:

• Modularity in xy-direction: They created dimers, trimers, tetramers, pentamers, and hexamers in a single-step reaction. with high yields (from 95% to 80%) and characterized them using agarose gel and electron microscopy.

• Modularity in z-direction: They successfully stacked structures vertically, creating dimers to pentamers with close to 100% efficiency and periodic tubes up to 5 μm in length!

Now that the researchers were confident that the connection systems worked, they started combining them in different ways:

• Finite superstructures: The authors used xy-direction and z-direction connections in the same monomers to create trimers, tetramers, pentamers, hexamers, and heptamers.

• Periodic superstructures: They also created DNA lattices spanning several micrometers!

• Parallel assembly: In this case, they leveraged the independence between the two connection systems to create different superstructures in the same reaction mixture, using distinct molecular triggers:

  • High Mg²⁺ concentration → xy-connections form.

  • Addition of connector strands → z-connections form.

• Selective disassembly: using molecular triggers such as ssDNA strands or changes in the salts concentration, the authors showed that the superstructures can be disassembled on command.

To conclude, this study presented a new method for the scalable and cost-effective assembly of higher order superstructures. I think this system has an edge when it comes to cost and simplicity: the ability to avoid purification is a big deal! At the moment, it is limited in how big structures can be created, and not all the monomer in all the structures can be individually addressed. But this is definitely a great step forward! There is more that I did not cover in the paper, so go and read it here!

And as always, thank you for reading! What are your thoughts on this paper and DNA origami? Reply and let me know.

In other news:

• Computing with DNA phages? What if you cold make phages only out of DNA, and then use them to compute? Well, if this was exactly your question, this paper has the answer. It introduces DNA nano-phage (DNP), a spatially confined molecular computing system that integrates nanobody-based recognition while minimizing off-target effects. It enables highly specific target cell detection in complex environments, advancing DNA-based diagnostics and immunotherapy.

• The importance of stacking: Of stacking DNA bases. This study shows that terminal stacking interactions significantly impact DNA nanostructure stability. By testing 16 stacking combinations in DNA tetrahedra, the researchers found that a single base stack can shift the melting temperature by up to 10°C. Strong stacking in a 4 bp sticky end enhances stability, making it comparable to a 6 bp weakly stacked end. An impressive amount of work!

• Shining light on DNA nanotubes: UV-light, to be precise. This study presents UV-responsive DNA nanotubes that self-assemble in a dose-dependent manner. By integrating RNA transcription, nanotube formation is controlled through both physical and biochemical stimuli. In synthetic cells, they mimic a stimulus-responsive cytoskeleton, offering new designs for dynamic biomolecular scaffolds. interesting for all the synthetic cells folks out there!

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