Vol 440 16 March 2006 doi: 10. 1038/nature04586 nature ARTICLES Folding dNa to create nanoscale shapes and patterns aul w, k rothemund 'Bottom-up fabrication which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. a key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down methods. the self-assembly of dna molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded dNa molecules into arbitrary two-dimensional shapes. the design for a desired shape is made by raster filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide'staple strands to hold the scaffold in place. Once synthesized and mixed the staple and scaffold strands self-assemble in a single step the resulting dNa structures are roughly 100 nm in diameter and approximate desired shapes such as squares disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles(which onstitutes a 30-megadalton molecular complex) In 1959, Richard Feynman put forward the challenge of writing the suggested that the folding of long strands could, in principle, proceed Encyclopaedia Britannica on the head of a pin, a task which he without many misfolding and avoid the problems of stoichiometry calculated would the use of dots 8 nm in size. Scanning probe and purification associated with methods that use many short DNA techniques have essentially answered this challenge: atomic force strands microscopy (AFM) and scanning tunnelling microscopy(STM) I now present a versatile and simple one-pot method for using allow us to manipulate individual atoms. But these techniques create numerous short single strands of DNA to direct the folding of a long, patterns serially(one line or one pixel at a time)and tend to require single strand of DNA into desired shapes that are roughly 100 nm in ultrahigh vacuum or cryogenic temperatures. As a result, methods diameter and have a spatial resolution of about 6 nm. I demonstrate ased on self-assembly are considered as promising alternatives that the generality of this method, which I term scaffolded DNA origami offer inexpensive, parallel synthesis of nanostructures under mild by assembling six different shapes, such as squares, triangles and five conditions. Indeed, the power of these methods has been demon- pointed stars. I show that the method not only provides access to strated in systems based on components ranging from porphyrins to structures that approximate the outline of any desired shape, but also whole viral particles. However, the ability of such systems to yield enables the creation of structures with arbitrarily shaped holes or structures of high complexity remains to be demonstrated. In surface patterns composed of more than 200 individual pixels. The particular, the difficulty of engineering diverse yet specific binding patterns on the 100-nm-sized DNA shapes thus have a complexity interactions means that most self-assembled structures contain just a that is tenfold higher than that of any previously self-assembled ew unique that may be addressed arbitrary pattern and comparable to that achieved using AFM and Nucleic acids can help overcome this problem: the exquisite STM surface manipulation specificity of Watson-Crick base pairing allows a combinatorially large set of nucleotide sequences to be used when designing binding Design of scaffolded DNA origam interactions. The field of DNA nanotechnology has exploited this The design of a DNA origami is performed in five steps, the first two property to create a number of more complex nanostructures, by hand and the last three aided by computer( details in Supplemen including two-dimensional arrays with 8-16 unique positions and tary Note S1). The first step is to build a geometric model of a dNA less than 20 nm spacing,I, as well as three-dimensional shapes such structure that will approximate the desired shape Figure la shows ar as a cube and truncated octahedron>. However, because the example shape(outlined in red)that is 33 nm wide and 35 nm tall. synthesis of such nanostructures involves interactions between a The shape is filled from top to bottom by an even number of parallel large number of short oligonucleotides, the yield of complete double helices, idealized as cylinders. The helices are cut to fit the structures is highly sensitive to stoichiometry(the relative ratios of shape in sequential pairs and are constrained to be anini dic arraea, strands). The synthesis of relatively complex structures was thus of turns in length. To hold the helices together, a periodic array of thought to require multiple reaction steps and purifications, with the crossovers(indicated in Fig. la as small blue crosses)is incorporated ultimate complexity of DNA nanostructures limited by necessarily these crossovers designate positions at which strands running along low yields. Recently, the controlled folding of a long single DNA one helix switch to an adjacent helix and continue there. The trand into an octahedron was reported", an approach that may be resulting model approximates the shape within one turn(3.6nm thought of as'single-stranded DNAorigami. The success of this work in the x-direction and roughly two helical widths(4 nm) in the 2006 Nature Publishing Group© 2006 Nature Publishing Group Folding DNA to create nanoscale shapes and patterns Paul W. K. Rothemund1 ‘Bottom-up fabrication’, which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by ‘top-down’ methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide ‘staple strands’ to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex). In 1959, Richard Feynman put forward the challenge of writing the Encyclopaedia Britannica on the head of a pin1 , a task which he calculated would require the use of dots 8 nm in size. Scanning probe techniques have essentially answered this challenge: atomic force microscopy2 (AFM) and scanning tunnelling microscopy3,4 (STM) allow us to manipulate individual atoms. But these techniques create patterns serially (one line or one pixel at a time) and tend to require ultrahigh vacuum or cryogenic temperatures. As a result, methods based on self-assembly are considered as promising alternatives that offer inexpensive, parallel synthesis of nanostructures under mild conditions5 . Indeed, the power of these methods has been demon￾strated in systems based on components ranging from porphyrins6 to whole viral particles7 . However, the ability of such systems to yield structures of high complexity remains to be demonstrated. In particular, the difficulty of engineering diverse yet specific binding interactions means that most self-assembled structures contain just a few unique positions that may be addressed as ‘pixels’. Nucleic acids can help overcome this problem: the exquisite specificity of Watson–Crick base pairing allows a combinatorially large set of nucleotide sequences to be used when designing binding interactions. The field of ‘DNA nanotechnology’8,9 has exploited this property to create a number of more complex nanostructures, including two-dimensional arrays with 8–16 unique positions and less than 20 nm spacing10,11, as well as three-dimensional shapes such as a cube12 and truncated octahedron13. However, because the synthesis of such nanostructures involves interactions between a large number of short oligonucleotides, the yield of complete structures is highly sensitive to stoichiometry (the relative ratios of strands). The synthesis of relatively complex structures was thus thought to require multiple reaction steps and purifications, with the ultimate complexity of DNA nanostructures limited by necessarily low yields. Recently, the controlled folding of a long single DNA strand into an octahedron was reported14, an approach that may be thought of as ‘single-stranded DNA origami’. The success of this work suggested that the folding of long strands could, in principle, proceed without many misfoldings and avoid the problems of stoichiometry and purification associated with methods that use many short DNA strands. I now present a versatile and simple ‘one-pot’ method for using numerous short single strands of DNA to direct the folding of a long, single strand of DNA into desired shapes that are roughly 100 nm in diameter and have a spatial resolution of about 6 nm. I demonstrate the generality of this method, which I term ‘scaffolded DNA origami’, by assembling six different shapes, such as squares, triangles and five￾pointed stars. I show that the method not only provides access to structures that approximate the outline of any desired shape, but also enables the creation of structures with arbitrarily shaped holes or surface patterns composed of more than 200 individual pixels. The patterns on the 100-nm-sized DNA shapes thus have a complexity that is tenfold higher than that of any previously self-assembled arbitrary pattern and comparable to that achieved using AFM and STM surface manipulation4 . Design of scaffolded DNA origami The design of a DNA origami is performed in five steps, the first two by hand and the last three aided by computer (details in Supplemen￾tary Note S1). The first step is to build a geometric model of a DNA structure that will approximate the desired shape. Figure 1a shows an example shape (outlined in red) that is 33 nm wide and 35 nm tall. The shape is filled from top to bottom by an even number of parallel double helices, idealized as cylinders. The helices are cut to fit the shape in sequential pairs and are constrained to be an integer number of turns in length. To hold the helices together, a periodic array of crossovers (indicated in Fig. 1a as small blue crosses) is incorporated; these crossovers designate positions at which strands running along one helix switch to an adjacent helix and continue there. The resulting model approximates the shape within one turn (3.6 nm) in the x-direction and roughly two helical widths (4 nm) in the ARTICLES 1 Departments of Computer Science and Computation & Neural Systems, California Institute of Technology, Pasadena, California 91125, USA. Vol 440|16 March 2006|doi:10.1038/nature04586 297