ARTICLES NATURETVol 440 16 March 2006 (Supplementary Note S8). Further, stocks of staples each contained a 2. Junno, T- Deppert, K, Montelius, L& Samuelson, L Controlled manipulation few per cent truncation products, stock concentrations were icles with an atomic force microscope. Appl. Phys. Lett. 66. 3627-362901995) measured with at least 10% error, and staples were used successfully 3. Eigler at stoichiometries that varied over an order of magnitude I suggest that several factors contribute to the success of scaffolded 4. Heinrich, A J, Lutz, C P, Gupta, J.A.& Eigler, D M Molecular cascades. DNA origami (even though the method ignores the normal, careful practices of DNA nanotechnology). These are(1)strand invasion,(2) 5. Whitesides, G M, Mathias, J. P. Seto, C T. Molecular self-assembly and anochemistry: a chemical strategy for the synthesis of nanostructures. Science an excess of staples,( 3)cooperative effects and(4)design that intentionally does not rely on binding between staples. Briefly 6. Yokoyama, T, Yokoyama, 5, Kamikado, T, Okuno, Y& Mashiko, S (details are given in Supplementary Note S9), strand invasion may elf-assembly on a surface of supramolecular aggregates with controlled size allow correct binding of excess full-length staples to displace 7. Mao, C.B.et al. Virus-based toolkit for the directed synthesis of magnetic and staples. Further, each correct addition of a staple organizes the 8. Seeman, N C Nucleic-acid junctions and lattices. I. Theor. Biol.99, 237-24 scaffold for subsequent binding of adjacent staples and precludes a (1982) large set of undesired secondary structures. Last, because staples are 9. Seeman, N.C. Lukeman, P. S. Nucleic acid nanostructures botton not designed to bind one another, their relative concentrations do 10. Chworos, A et al. Building programmable jigsaw puzzles with RNA. Science not matter 306,2068-2072(2004) The method presented here is easy to implement, high yield and 1. Park, S. H et al. Finite-size, fully-addressable DNA tile relatively inexpensive. Three months of effort went into the design 12. Chen, 1. Seeman, N. C. The synthesis from DNA of a molecule with the design and one week to synthesize( commercially); the mixing and 13. Zhang, Y. Seeman, N. C. The construction of a DNA truncated octahedron annealing of strands required a few hours. The greatest experimental J. Am. chen.Soc16.1661-16690199 difficulty was acquiring high-resolution AFM images, typically 14. Shih, WM, Quispe, J.D.& Joyce, G.F. A1.7-kilobase single-stranded DNA taking two days per structure. For rigid designs using circular that folds into a nanoscale octahedron Nature 427, 618-621(2004) 15. Rothemund, P. w.K et al. Design and characterization of programmable dNa scaffolds(rectangles with patterns, three-hole disks, and sharp nanotubes. J Am. Chem. Soc. 26, 16344-16353(2004) triangles), yields of qualitatively well-formed structures were at 16. Fu, T-J.& Seeman, N C DNA double-crossover molecules. Biochemistry 32, destructive imaging and quantification of small (<15 nm)defects. A 17. LaBean, T H, infree, E. Reif, J.H. in DNA Based Computers V(eds Winfree, possible objection to the routine use of the method is the potential Rhode Island, 1999) E& Gifford, D K )123-140( Vol 54 of DIMACS, AMS Press, Providence cost of staples; unlike the scaffold, staples cannot be cloned. However, 18. Yan, H, LaBean, T.H., Feng. L& Reif, J H Directed nucleation assembly of do purified strands are inexpensive so that the scaffold constitutes DNA tile compl or barcode-patterned lattices. Proc Natl Acad. Sci. USA 0% of the cost, even when using a 100-fold excess of staples 0,8103-8108(2003 (Supplementary Note S10) 19. Reif, J H. in Proc. 29th Int Colloquium on Automata, Languages, and Programming (CALP)(eds Widmayer, P. Ruiz, F T Bueno, R. m, Hennessy, M, Eidenben I believe that scaffolded DNA origami can be adapted to create S& Conejo, R )1-21(Vol. 2380 of Lecture Notes in Computer Science, more complex or larger structures. For example, the design of three dimensional structures should be accessible using a straightforward 20. Seeman, N C De novo design of sequences for nucleic acid structural adaptation of the raster fill method given here. If non-repetitive 21. winf ed Computers(eds Lipton, R J. Baum, E. B )199-221 origami with 20,000 features may be possible. However, the require. 22. Rothemund p.K. AMS Press, providence, Rhode Island, 1996) Algorithmic self-assembly of ment for unique sequence information means that the method NA Sierpinski triangles. PLos Biol. 2, e424 (2004) cannot be scaled up arbitrarily; whenever structures above a critical 23. Yan, H, Park, 5. H, Finkelstein, G,Reif, JH&LaBean,THDNA-templated size or level of complexity are desired, it will therefore be necessary oly of protein arrays and highly conductive nanowires. Science 301, to combine scaffolded DNA origami with hierarchical self- 24. Le, J.D. et al DNA-templated self-assembly of metallic nanocomponent arrays algorithmic self-assembly or top-down fabrication on a surface Nano Lett 4, 2343-2347(2004) techniques creationofananobreadboard,towhichdiversecomponentscouldwww.nature.com/naturcnislinkedtotheonlineversionofthepaperat be added. The attachment of proteins"), for example, might allow Acknowledgements I thank E Winfree for discussions and providing a assemblies and examining the effects of spatial organization, whereas N. Papadakis, L. Adleman, 1. Goto, R. Barish, R. Schulman, R. Hariadi, M. Cook novel biological experiments aimed at modelling complex protein N attaching nanowires, carbon nanotubes or gold nanoparticles"dbboooM. Diehl for discussions: B. Shaw for a gift of AFM tips: A Schmidt for molecular electronic or plasmonic circuits it be created by These ideas suggest that scaffolded DNA origami could find use in Foundation Career and Nano grants to E. Winfree as well as fellowships from Center f fields as diverse as molecular biology and device physics Author Information Reprints and permissions information is available at npg nature. com/reprintsandpermissions. The author declares competin financialinterestsdetailsaccompanythepaperonwww.nature.com/nature 1. Feynman, R. P. There' s plenty of room at the bottom. Engineering and Science equests for materials should be addressed to P.w.K.R. 23(5), 22-36(Caltech, February, 1960) (pwr@dna. caltech. edu) 2006 Nature Publishing Group© 2006 Nature Publishing Group (Supplementary Note S8). Further, stocks of staples each contained a few per cent truncation products, stock concentrations were measured with at least 10% error, and staples were used successfully at stoichiometries that varied over an order of magnitude. I suggest that several factors contribute to the success of scaffolded DNA origami (even though the method ignores the normal, careful practices of DNA nanotechnology). These are (1) strand invasion, (2) an excess of staples, (3) cooperative effects and (4) design that intentionally does not rely on binding between staples. Briefly (details are given in Supplementary Note S9), strand invasion may allow correct binding of excess full-length staples to displace unwanted secondary structure, incorrect staples, or grossly truncated staples. Further, each correct addition of a staple organizes the scaffold for subsequent binding of adjacent staples and precludes a large set of undesired secondary structures. Last, because staples are not designed to bind one another, their relative concentrations do not matter. The method presented here is easy to implement, high yield and relatively inexpensive. Three months of effort went into the design program. In addition, each structure required about one week to design and one week to synthesize (commercially); the mixing and annealing of strands required a few hours. The greatest experimental difficulty was acquiring high-resolution AFM images, typically taking two days per structure. For rigid designs using circular scaffolds (rectangles with patterns, three-hole disks, and sharp triangles), yields of qualitatively well-formed structures were at least 70%. A better understanding of folding will depend on less￾destructive imaging and quantification of small (,15 nm) defects. A possible objection to the routine use of the method is the potential cost of staples; unlike the scaffold, staples cannot be cloned. However, unpurified strands are inexpensive so that the scaffold constitutes 80% of the cost, even when using a 100-fold excess of staples (Supplementary Note S10). I believe that scaffolded DNA origami can be adapted to create more complex or larger structures. For example, the design of three￾dimensional structures should be accessible using a straightforward adaptation of the raster fill method given here. If non-repetitive scaffolds of megabase length can be prepared, micrometre-size origami with 20,000 features may be possible. However, the require￾ment for unique sequence information means that the method cannot be scaled up arbitrarily; whenever structures above a critical size or level of complexity are desired, it will therefore be necessary to combine scaffolded DNA origami with hierarchical self￾assembly10,11, algorithmic self-assembly22, or top-down fabrication techniques. An obvious application of patterned DNA origami would be the creation of a ‘nanobreadboard’, to which diverse components could be added. The attachment of proteins23, for example, might allow novel biological experiments aimed at modelling complex protein assemblies and examining the effects of spatial organization, whereas molecular electronic or plasmonic circuits might be created by attaching nanowires, carbon nanotubes or gold nanoparticles24. These ideas suggest that scaffolded DNA origami could find use in fields as diverse as molecular biology and device physics. Received 7 September 2005; accepted 12 January 2006. 1. Feynman, R. P. There’s plenty of room at the bottom. Engineering and Science 23(5), 22–-36 (Caltech, February, 1960). 2. Junno, T., Deppert, K., Montelius, L. & Samuelson, L. Controlled manipulation of nanoparticles with an atomic force microscope. Appl. Phys. Lett. 66, 3627–-3629 (1995). 3. Eigler, D. M. & Schweizer, E. K. Positioning single atoms with a scanning tunnelling microscope. Nature 344, 524–-526 (1990). 4. Heinrich, A. J., Lutz, C. P., Gupta, J. A. & Eigler, D. M. Molecular cascades. Science 298, 1381–-1387 (2002). 5. Whitesides, G. M., Mathias, J. P. & Seto, C. T. Molecular self-assembly and nanochemistry: a chemical strategy for the synthesis of nanostructures. Science 254, 1312–-1319 (1991). 6. Yokoyama, T., Yokoyama, S., Kamikado, T., Okuno, Y. & Mashiko, S. Self-assembly on a surface of supramolecular aggregates with controlled size and shape. Nature 413, 619–-621 (2001). 7. Mao, C. B. et al. Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires. Science 303, 213–-217 (2004). 8. Seeman, N. C. Nucleic-acid junctions and lattices. J. Theor. Biol. 99, 237–-247 (1982). 9. Seeman, N. C. & Lukeman, P. S. Nucleic acid nanostructures: bottom-up control of geometry on the nanoscale. Rep. Prog. Phys. 68, 237–-270 (2005). 10. Chworos, A. et al. Building programmable jigsaw puzzles with RNA. Science 306, 2068–-2072 (2004). 11. Park, S. H. et al. Finite-size, fully-addressable DNA tile lattices formed by hierarchical assembly procedures. Angew. Chem. 118, 749–-753 (2006). 12. Chen, J. & Seeman, N. C. The synthesis from DNA of a molecule with the connectivity of a cube. Nature 350, 631–-633 (1991). 13. Zhang, Y. & Seeman, N. C. The construction of a DNA truncated octahedron. J. Am. Chem. Soc. 116, 1661–-1669 (1994). 14. Shih, W. M., Quispe, J. D. & Joyce, G. F. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618–-621 (2004). 15. Rothemund, P. W. K. et al. Design and characterization of programmable DNA nanotubes. J. Am. Chem. Soc. 26, 16344–-16353 (2004). 16. Fu, T.-J. & Seeman, N. C. DNA double-crossover molecules. Biochemistry 32, 3211–-3220 (1993). 17. LaBean, T. H., Winfree, E. & Reif, J. H. in DNA Based Computers V (eds Winfree, E. & Gifford, D. K.) 123–-140 ( Vol. 54 of DIMACS, AMS Press, Providence, Rhode Island, 1999). 18. Yan, H., LaBean, T. H., Feng, L. & Reif, J. H. Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices. Proc. Natl Acad. Sci. USA 100, 8103–-8108 (2003). 19. Reif, J. H. in Proc. 29th Int. Colloquium on Automata, Languages, and Programming (ICALP) (eds Widmayer, P., Ruiz, F. T., Bueno, R. M., Hennessy, M., Eidenbenz, S. & Conejo, R.) 1–-21 (Vol. 2380 of Lecture Notes in Computer Science, Springer, New York, 2002). 20. Seeman, N. C. De novo design of sequences for nucleic acid structural engineering. J. Biomol. Struct. Dyn. 8, 573–-581 (1990). 21. Winfree, E. in DNA Based Computers (eds Lipton, R. J. & Baum, E. B.) 199–-221 (Vol. 27 of DIMACS, AMS Press, Providence, Rhode Island, 1996). 22. Rothemund, P. W. K., Papadakis, N. & Winfree, E. Algorithmic self-assembly of DNA Sierpinski triangles. PLoS Biol. 2, e424 (2004). 23. Yan, H., Park, S. H., Finkelstein, G., Reif, J. H. & LaBean, T. H. DNA-templated self-assembly of protein arrays and highly conductive nanowires. Science 301, 1882–-1884 (2003). 24. Le, J. D. et al. DNA-templated self-assembly of metallic nanocomponent arrays on a surface. Nano Lett. 4, 2343–-2347 (2004). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements I thank E. Winfree for discussions and providing a stimulating laboratory environment; B. Yurke for the term ‘nanobreadboard’; N. Papadakis, L. Adleman, J. Goto, R. Barish, R. Schulman, R. Hariadi, M. Cook and M. Diehl for discussions; B. Shaw for a gift of AFM tips; A. Schmidt for coordinating DNA synthesis; and K. Yong, J. Crouch and L. Hein for administrative support. This work was supported by National Science Foundation Career and Nano grants to E. Winfree as well as fellowships from the Beckman Foundation and Caltech Center for the Physics of Information. Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The author declares competing financial interests: details accompany the paper on www.nature.com/nature. Correspondence and requests for materials should be addressed to P.W.K.R. (pwkr@dna.caltech.edu). ARTICLES NATURE|Vol 440|16 March 2006 302