Translation of DNA Signals into polymers assembly instructions

Translation of DNA Signals into polymers assembly instructions

(Parte 1 de 2)

DOI: 10.1126/science.1104299 , 2072 (2004); 306Science et al.Shiping Liao, Instructions Translation of DNA Signals into Polymer Assembly

w.sciencemag.org (this information is current as of July 24, 2009 ): The following resources related to this article are available online at http://www.sciencemag.org/cgi/content/full/306/5704/2072 version of this article at: including high-resolution figures, can be found in the onlineUpdated information and services, http://www.sciencemag.org/cgi/content/full/306/5704/2072/DC1 can be found at: Supporting Online Material found at: can berelated to this articleA list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/306/5704/2072#related-content 72 article(s) on the ISI Web of Science. cited byThis article has been http://www.sciencemag.org/cgi/content/full/306/5704/2072#otherarticles 5 articles hosted by HighWire Press; see: cited byThis article has been http://www.sciencemag.org/cgi/collection/chemistry Chemistry : subject collectionsThis article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprintsInformation about obtaining registered trademark of AAAS. is aScience2004 by the American Association for the Advancement of Science; all rights reserved. The title CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience on July 24, 2009 w.sciencemag.org Downloaded from as Kd values measured for single 3¶ tail connectors are in the micromolar range. This fact and the small number of overlapping RNA lattices observed by AFM suggest that epitaxial phenomena occurrin g between RNAs and magnesium ions adsorbed on the negative mica surface might promote assembly. The observed RNA networks grow in a radial fashion. All LT tectosquare arrays involve a similar number of molecules, indicating that LT assembly is independent of the nature of the pattern formed (Fig. 3). However, STs generate significantly larger lattice networks. Despite their apparent robustness and stiffness, multiple AFM scans can disrupt the edges or deplete one or two tectosquares within the lattice. It is clear that, as a soft matter medium, the stability and size of RNA networks can still be improved and their visualization by AFM still remains challenging.

This work offers an attractive alternative to DNA, protein, and synthetic molecules for directed arrangement of matter at a molecular level (1, 30–3) and lays the foundation for generalization to periodic 3D nanomaterials of RNA. As fully addressable, programmable assemblies, tectosquare jigsaw puzzles can serve as hosts to organize at relative defined positions various molecular components with high precision and to generate nanochips, nanocircuits, and nanocrystals with potential applications in nanotechnology and material sciences (1, 32). With its underlying modular and hierarchical construction displaying a minimal set of primitive operations, the tectosquare system could possibly be a Turing-universal computing molecular system (10, 34, 35). It can also be a valuable tool for studying self-organization and emergence of complexity out of randomness (35). For instance, an unanswered question is whether combinatorial population of tectosquares could still assemble accurately into organized architectures.

Nature 394, 539 (1998). 5. H. Yan, S. H. Park, G. Finkelstein, J. H. Reif, T. H.

LaBean, Science 301, 1882 (2003). 6. H. Yan, T. H. LaBean, L. Feng, J. H. Reif, Proc. Natl.

Acad. Sci. U.S.A. 100, 8103 (2003). 7. C. Mao, W. Sun, Z. Shen, N. C. Seeman, Nature 397, 144 (1999). 8. B. Yurke, A. J. Turberfield, A. P. Mills Jr., F. C. Simmel,

J. L. Neumann, Nature 406, 605 (2000). 9. H. Yan, X. Zhang, Z. Shen, N. C. Seeman, Nature 415, 62 (2002). 10. E. Winfree, J. Biomol. Struct. Dyn. Conversat. 1, 263 (2000). 1. R. T. Batey, R. P. Rambo, J. A. Doudna, Angew. Chem.

Int. Ed. Engl. 38, 2326 (1999). 12. N. Ban, P. Nissen, J. Hansen, P. B. Moore, T. A. Steitz,

15. E. Westhof, B. Masquida, L. Jaeger, Fold. Des. 1, R78 (1996). 16. L. Jaeger, N. B. Leontis, Angew. Chem. Int. Ed. Engl. 39, 2521 (2000). 17. L. Jaeger, E. Westhof, N. B. Leontis, Nucleic Acids Res. 29, 455 (2001). 18. Y. Ikawa, K. Fukada, S. Watanabe, H. Shiraishi, T. Inoue,

Structure (Cambridge) 10, 527 (2002). 19. S. Horiya et al., Chem. Biol. 10, 645 (2003). 20. B. Liu, S. Baudrey, L. Jaeger, G. C. Bazan, J. Am. Chem.

Soc. 126, 4076 (2004). 21. Materials and methods are available as supporting material on Science Online. 2. E. Ennifar, P. Walter, B. Ehresmann, C. Ehresmann,

P. Dumas, Nature Struct. Biol. 8, 1064 (2001). 23. Considering that each of the four tectosquare unitscan either have no tail, or a 3¶ tail of n different sequences with different size (s) and orientation (o), a total of 4(son þ 1) different a, b, c, and d tectoRNAs can be combinedto construct(son þ 1)4 tectosquares. 24. R. M. Dirks, M. Lin, E. Winfree, N. A. Pierce, Nucleic

Acids Res. 32, 1392 (2004). 25. A. A. Szewczak, E. R. Podell, P. C. Bevilacqua, T. R.

Cech, Biochemistry 37, 11162 (1998). 26. J. S. Lodmell, C. Ehresmann, B. Ehresmann, R. Marquet,

J. Microsc. 212, 273 (2003). 28. LTs associate better when tails are oriented parallel to their a-d and b-c sides. This fact supports our twofold symmetrical models (Fig. 1C; fig. S1), with a-d and b-c being parallel to each other and a-b and c-d being tilted. Assembly through tails oriented parallel to a-b and c-d is nevertheless possible. 29. With 12 different tail-tail connectors, we are pres- ently able to generate a 3 by 3 grid made of nine different tectosquares with the position of each of the 36 constitutive tectoRNAs being fully addressable within the RNA lattice. 30. J. Michl, T. F. Magnera, Proc. Natl. Acad. Sci. U.S.A. 9, 4788 (2002). 31. N. C. Seeman, A. M. Belcher, Proc. Natl. Acad. Sci.

U.S.A. 9 (suppl. 2), 6451 (2002). 32. S. Zhang, Nature Biotechnol. 21, 1171 (2003). 3. P. Ringler, G. E. Schulz, Science 302, 106 (2003). 34. A. Carbone, N. C. Seeman, Proc. Natl. Acad. Sci. U.S.A. 9, 12577 (2002). 35. S. Wolfram, A New Kind of Science (Wolfram Media,

Champaign, IL, 2002). 36. This paper is dedicated to St. Joseph, patron saint of carpenters,and to F. Michel and E. Westhof,L.J.’s mentors.Thanksto S. and L. Baudrey for technicalassistance and to C. Geary, H. Waite, and S. Parsons for critical reading of the manuscript. A.C. thanks the Department of Bioorganic Chemistry, Polish Academy of Sciences, CM&MS, Lodz, 90363, Poland. Funding for this work was provided by faculty start-up funds from UCSB to L.J. and by grants from NSF to L.J. (CHE-0317154 and MRSEC DMR00-80034) and H.H. (MCB0236093).

Supporting Online Material w.sciencemag.org/cgi/content/full/306/5704/2068/ DC1 Materials and Methods Figs. S1 to S4 Tables S1 to S3 References and Notes

31 August 2004; accepted 8 November 2004 10.1126/science.1104686

Translation of DNA Signals into

Polymer Assembly Instructions

Shiping Liao and Nadrian C. Seeman*

We developed a DNA nanomechanical device that enables the positional synthesis of products whose sequences are determined by the state of the device. This machine emulates the translational capabilities of the ribosome. The device has been prototyped to make specific DNA sequences. The state of the device is established by the addition of DNA set strands. There is no transcriptional relationship between the set strands and the product strands. The device has potential applications that include designer polymer synthesis, encryption of information, and use as a variable-input device for DNA-based computation.

We built a DNA nanomechanical device that mimics the translational capabilities of the ribosome. In response to a DNA signal, it aligns a series of molecules in specific positions; these molecules are then fused together in a specific order. For convenience, we have prototyped this system with DNA, so the products are DNA oligonucleotides of a defined sequence. Thus, in this case, the chemistry of the product is similar to that of the signal molecules, but there is no complementary relationship to the signal sequences. By using DNA molecules to set the states of two DNA grammed the synthesis of four different product molecules.

ThePX-JX2 deviceis a sequence-dependent

DNA machine, the state of which is controlled by hybridization topology (1). It can assume two structural states (termed PX and JX2 ), which differ from each other by a half-turn rotation of one end of the molecule relative to the other end (Fig. 1A). Two different pairs of set strands can bind to the framework of the device, thereby establishing which structural state it adopts. The set strands contain short unpaired segments (Btoeholds[) at one end to facilitate their removal by unset strands that bind to the toeholds and then remove the set strands by branch migration (2).

In addition to the PX-JX2 device, numerous variants of sequence-dependent control, pioneered in DNA tweezers by Yurke et al.( 2), have been reported; these include a DNA

Department of Chemistry, New York University, New York, NY 10003, USA.

*To whom correspondence should be addressed. E-mail: ned.seeman@nyu.edu

17 DECEMBER 2004 VOL 306 SCIENCE w.sciencemag.org2072 on July 24, 2009 w.sciencemag.org Downloaded from actuator (3), a three-state device (4), and a DNA bipedal walking machine (5). Neither these nor other shape-shifting DNA devices (6) have been incorporated into a larger context that performs a useful task. By contrast, we used the structural state of the device reported here for positional control of the products of polymer concatenation.

We incorporated two PX-JX2 devices in succession, thereby controlling the relative orientations of a diamond-shaped motif (7) and a pair of double-diamond-shaped wings (Fig. 1B). The Arabic numerals in Fig. 1B label individual sticky ends; these sticky ends are available to bind DNA double-crossover (DX) molecules (8) that contain a continuous DNA strand extending from one end to the other. This continuous strand ultimately will be a component of the product. The rest of the DX molecule plays a role analogous to that of tRNA in translation: It serves as an adaptor between the strand that it carries and the device. The set strands of the device contain the signal, or message, that configures the sticky ends to bind one of a pair of DX molecules in each of the two gaps.

Figure 1C illustrates the flow chart for the experiments we performed. First, the device is constructed with both PX-JX 2 machines in the PX state. Unset strands,

Fig. 1. Schematic drawings of the system and its components. (A) The PX state of the device is shown at the left, with green set strands. These are removed by biotinylated (black dots) unset strands to leave a naked frame (top). Adding the purple set strands puts the device in the JX2 state. The bottom of the cycle shows restoration of the PX state. The two states differ by a half-turn rotation, as highlighted by the letters A, B, C, and D flanking the helices. (B) Five diamond motifs (7) are labeled by Roman numerals, and the sticky ends are labeled by Arabic numerals. The diamonds are connected to form double-diamond wings by means of a PX linkage, to give the wings dyad symmetry (14). There is an initiator-diamond motif at the left (I), and two doublediamond motifs are at the center and right. The initiator diamond and the double diamonds are connected by PX-JX2 devices, so the relative orientations of the sticky ends can be varied. (C) The initial assembly of the device is shown at the top, similar to the structure in (B). Diamonds I and

IV have been drawn with different colors for clarity. A and B indicate sticky ends used in purification. The next step entails setting the state of the device. The DX molecules are all added to the solution, along with an initiator DX1 (black) and a biotinylated double strand, DS1, complementary to A. After left-side purification by magnetic streptavidin (MS) beads, and release by cleaving DS1, right-side purification is achieved similarly with DS2 and the addition of magnetic beads again. The purified complex is then ligated, and the continuous strand is denatured, purified, and sequenced. (D) The four ligated DX molecules are shown with the same color coding as in (C). The continuous strands across the top are the final products that are sequenced.

Fig. 2. (A) This is a 3.5% nondenaturing gel of the complex, composed of all 2 strands. LM indicates linear markers. Each of the strands [numbering in (9) and figs. S1 to S3] is labeled separately with radioactive phosphate. The number above each lane shows the labeled strand. In each case, the strand is incorporated cleanly into the complex, with no doubling and no breakdown products evident. (B) The AFM images were prepared as described previously (1). The DX molecules were omitted for clarity in the image. The left image is a large field, and the right image is a zoom. The single- and double-diamond nature of the complex is evident from the small-large-large nature of the sample molecules. It is clear that the purification protocol used is quite successful at eliminating failure products from the sample.

Fig. 3. This is a 6% denaturing gel autoradiogram showing the products of the ligation. The first lane contains a 50-nucleotide ladder. Lanes 2 to 5 contain, respectively, the products of setting the device states to PX-PX, PX-JX2,

JX2-PX, and JX2-JX2. The black strand (Fig. 1D) is labeled in each case. The target band is prominent in all lanes, although failure products are evident. The band between 150 and 200 may be an erroneous product; it represents 0.3%, 0.2%, 1.0%, and 1.2%, of the material in lanes 2 to 5, respectively.

w.sciencemag.org SCIENCE VOL 306 17 DECEMBER 2004 2073 on July 24, 2009 w.sciencemag.org Downloaded from followed by specific set strands, are then added to the device, setting its state. The complete set of DX molecules is added to the solution, and the correct ones (cyan and green in Fig. 1C) bind in the correct sites between the diamond structures, as dictated by the sticky ends. In addition, an Binitiator[ DX (black in Fig. 1C) is bound on the top of the leftmost double diamond. After ligation, the DX molecules are dissociated, the target strand is isolated, and its sequence is determined. Its target length is longer than any other strand in the system, so it is easy to isolate from failure products and fortuitous molecules that bind to the opposite (bottom) side of the device. Figure 1D shows the way that the selection of set strands directs the synthesis of different products, in the same way that different mRNA molecules direct the synthesis of different polypeptide chains. Experimental methods are described in (9).

(Parte 1 de 2)

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