Scientists at the California Institute of Technology (Caltech) have created what is possibly the world’s smallest Mona Lisa, using a mosaic of individual tiles of folded strands of DNA that, when viewed under atomic force microscopy (AFM), appears unmistakably as the enigmatic face of Leonardo da Vinci’s famous painting. The technique, developed by researchers in the laboratory of assistant professor of bioengineering Lulu Qian, Ph.D., builds on the original DNA origami technology developed by Caltech’s Paul Rothemund, Ph.D., but allows the construction of much larger mosaics, or arrays. The Caltech researchers project that the method could one day enable scientists to build complex components in DNA robots, localized DNA circuits, and nanostructures to help study molecular interactions in chemical and biological systems.

They describe their technology in Nature, in a paper entitled “Fractal Assembly of Micrometre-Scale DNA Origami Arrays with Arbitrary Patterns.”

Making a single tile, or square, of DNA origami involves taking one long stand of DNA and a number of shorter strands, or DNA staples. Add all the pieces together in a test tube and the short strands bind to specified sites on the long strand, causing it to fold into a particular shape. The Caltech team has developed a method for self-assembling these individual squares into a larger DNA “canvas,” or array, to make up a desired picture. Attaching molecules selectively to the staples creates a raised pattern that can then be viewed by AFM, revealing the picture.

The original DNA origami technique developed by Dr. Rothemund—who is currently Caltech research professor of bioengineering, computing, and mathematical sciences, and computation and neural systems—was reported back in 2006. It allowed scientists to create self-assembling DNA structures into patterns, such as a smiley face, but size was a limiting factor. It wasn’t possible to create nanostructures of more that about 0.05 μm2, Dr. Qian’s team reports.

The Caltech researchers have now developed a hierarchical, multistage assembly process that allows them to create much larger DNA origami arrays. The approach involves dividing the complete picture into individual square sections and then determining which DNA sequence is needed to make up each square. The challenge for the Caltech team was how to get the individual squares to self-assemble into the complete picture. “We could make each tile with unique edge staples so that they could only bind to certain other tiles and self-assemble into a unique position in the superstructure,” explains Grigory Tikhomirov, Ph.D., the paper's lead author, “but then we would have to have hundreds of unique edges, which would be not only very difficult to design but also extremely expensive to synthesize. We wanted to only use a small number of different edge staples but still get all the tiles in the right places.”

The team addressed this by assembling the tiles in stages, a bit like making up small sections of a jigsaw puzzle. You first piece individual tiles together into small sections of the puzzle, then fit the small sections together to make up larger sections, and then put the larger sections together to complete the puzzle. For the fractal assembly technology each mini-puzzle has the same four edges, but because each section is assembled separately, there’s no risk that individual pieces will attach in the wrong place. The team has called the method “fractal assembly” because each stage of construction uses the same set of assembly rules. 

Importantly, the hierarchical nature of the method means that it requires only a small, constant set of DNA strands with unique sequences to build structures with increasing sizes and a potentially limitless library of pictures, suggests Dr. Tikhomirov. “This economical approach of building more with less is similar to how our bodies are built.”

“Once we have synthesized each individual tile, we place each one into its own test tube for a total of 64 tubes,” continues Philip Petersen, the paper's co-first author. “We know exactly which tiles are in which tubes, so we know how to combine them to assemble the final product. First, we combine the contents of four particular tubes together until we get 16 two-by-two squares. Then those are combined in a certain way to get four tubes each with a four-by-four square. And then the final four tubes are combined to create one large, eight-by-eight square composed of 64 tiles. We design the edges of each tile so that we know exactly how they will combine.”

The team used this fractal assembly approach to generate DNA origami arrays with sizes of up to 0.5   μm2, and with up to 8704 individual pixels. Their Mona Lisa is about 64 times larger than the original DNA origami structures designed by Dr. Rothemund, but, because the method effectively recycles the same edge interactions at each construction stage, the number of different DNA strands required for the assembly of the complete structure is about the same as for Rothemund's original origami. This should make the overall process more cost effective, Qian suggests.

The researchers want to make their technique accessible to other researchers, and have developed a software tool that converts a desired image into DNA strands, and also generates the required wet-lab protocols. “The protocol can be directly read by a liquid-handling robot to automatically mix the DNA strands together,” Dr. Qian states. “The DNA nanostructure can be assembled effortlessly.” As well as creating the Mona Lisa, the researchers have used the technology to generate a bacterium-sized image of a rooster and a life-sized picture of a bacterium.

“Other researchers have previously worked on attaching diverse molecules such as polymers, proteins, and nanoparticles to much smaller DNA canvases for the purpose of building electronic circuits with tiny features, fabricating advanced materials, or studying the interactions between chemicals or biomolecules,” Petersen notes. “Our work gives them an even larger canvas to draw upon.”

The authors suggest that, in principle, the DNA nanostructures are large enough to be integrated directly into top-down, high-throughput, low-cost manufacturing techniques, such as photolithography, which could feasibly enable the construction of complex, nanoscale-featured devices. Future developments could also improve the fractal assembly method further and allow scientists to generate even larger origami arrays, or even three-dimensional structures.

Nevertheless, they conclude, “Even without such improvements, fractal assembly makes it possible to scale up the complexity of DNA nanostructures with arbitrary patterns, using a simple algorithm and a constant number of unique strands.… When used in conjunction with a software tool that we developed that converts an arbitrary pattern into DNA sequences and experimental protocols, our assembly method is readily accessible and will facilitate the construction of sophisticated materials and devices with sizes similar to that of a bacterium using DNA nanostructures.”


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