Caltech Team Manages to “Paint” Smallest Mona Lisa Using DNA

Caltech's Paul Rothemund (BS '94)—currently research professor of bioengineering, computing and mathematical sciences, and computation and neural systems—developed a technique to fold a long strand of DNA into a prescribed shape way back in 2006. The method, labeled DNA origami, enabled researchers to develop self-assembling DNA structures that could take on any specified pattern, such as a 100-nanometer-wide smiley face.

The process of fractal assembly, using wooden puzzle pieces. (Credit: Caltech)

DNA origami transformed the field of nanotechnology, paving way to possibilities of building minute molecular devices or "smart" programmable materials. However, a few of these applications require a lot larger DNA origami structures.

Now, researchers in the laboratory of Lulu Qian, assistant professor of bioengineering at Caltech, have developed an economical technique by which DNA origami self-assembles into large arrays with totally customizable patterns, forming a sort of canvas that can exhibit any image. To show this, the team created the world's smallest recreation of Leonardo da Vinci's Mona Lisa—using DNA.

The study is described in a paper published in the December 7 issue of the journal Nature.

While DNA is mostly well known for encoding the genetic information of living things, the molecule is also an exceptional chemical building block. A single-stranded DNA molecule is made up of smaller molecules known as nucleotides—abbreviated A, T, C, and G—organized in a string, or sequence. The nucleotides in a single-stranded DNA molecule can bond with those of another single strand to create double-stranded DNA, but the nucleotides bind only in very particular ways: a C nucleotide with a G or an A nucleotide with a T. These stringent base-pairing "rules" render it possible to design DNA origami.

To create a single square of DNA origami, one only requires a long single strand of DNA and numerous shorter single strands—known as staples—designed to bind to several designated places on the long strand. When the short staples and the long strand are united in a test tube, the staples pull regions of the long strand together, causing it to fold over itself into the anticipated shape. A large DNA canvas is formed out of many smaller square origami tiles, like assembling a puzzle. Molecules can be selectively attached to the staples so as to create a raised pattern that can be viewed using atomic force microscopy.

The Caltech team created software that can use an image such as the Mona Lisa, split it up into small square sections, and establish the DNA sequences required to make up those squares. Next, their test was to make those sections self-assemble into a superstructure that recreates the Mona Lisa.

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,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.

Grigory Tikhomirov, senior postdoctoral scholar and the paper's lead author

The strategic part in achieving this was to assemble the tiles in stages, like putting together small regions of a puzzle and then assembling those to make larger regions before finally putting the larger regions together to make the finished puzzle. Each tiny puzzle utilizes the same four edges, but because these puzzles are assembled independently, there is no risk, for instance, of a corner tile attaching in the incorrect corner. The team has termed the technique "fractal assembly" because the same set of assembly rules is applied at varying scales.

Once we have synthesized each individual tile, we place each one into its own test tube for a total of 64 tubes, 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.

Philip Petersen, graduate student, co-first author on the paper.

The Qian team's final structure was 64 times larger than the innovative DNA origami structure created by Rothemund in 2006. Extraordinarily, because of the recycling of the same edge interactions, the number of different DNA strands necessary for the assembly of this DNA superstructure was approximately the same as for Rothemund's original origami. This should make the new technique similarly economical, according to Qian.

The hierarchical nature of our approach allows using only a small and constant set of unique building blocks, in this case DNA strands with unique sequences, to build structures with increasing sizes and, in principle, an unlimited number of different paintings, this economical approach of building more with less is similar to how our bodies are built. All our cells have the same genome and are built using the same set of building blocks, such as amino acids, carbohydrates, and lipids. However, via varying gene expression, each cell uses the same building blocks to build different machinery, for example, muscle cells and cells in the retina.

Grigory Tikhomirov, senior postdoctoral scholar and the paper's lead author

The team also developed software to enable scientists everywhere to produce DNA nanostructures using fractal assembly.

"To make our technique readily accessible to other researchers who are interested in exploring applications using micrometer-scale flat DNA nanostructures, we developed an online software tool that converts the user's desired image to DNA strands and wet-lab protocols," says Qian. "The protocol can be directly read by a liquid-handling robot to automatically mix the DNA strands together. The DNA nanostructure can be assembled effortlessly."

Employing this online software tool and automatic liquid-handling methods, several other patterns were designed and put together using DNA strands, including a bacterium-sized portrait of a rooster and a life-sized portrait 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, our work gives them an even larger canvas to draw upon.

Philip Petersen, graduate student, co-first author on the paper.

The research paper is titled "Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns." The study was funded by the Burroughs Wellcome Fund, the National Institutes of Health's National Research Service Award, and the National Science Foundation.

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