Operation of a DNA Robot Arm Inserted into a 2D DNA

Operation of a DNA Robot Arm Inserted into a 2D DNA

DOI: 10.1126/science.1131372 , 1583 (2006); 314Science et al.Baoquan Ding, DNA Crystalline Substrate Operation of a DNA Robot Arm Inserted into a 2D

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R. Astron. Soc. 274, 964 (1995). 23. We are indebted to R. Palasek for managing the code and for assistance with the graphics. This study has been carried out under the auspices of the U.S. Department of Energy, National Nuclear Security Administration, by the University of California, Lawrence Livermore National Laboratory, under contract W-7405-Eng-48.

Supporting Online Material w.sciencemag.org/cgi/content/full/1133065/DC1 Movie S1

26 July 2006; accepted 12 October 2006 Published online 26 October 2006; 10.1126/science.1133065 Include this information when citing this paper.

Operation of a DNA Robot Arm Inserted into a 2D DNA

Crystalline Substrate Baoquan Ding and Nadrian C. Seeman*

The success of nanorobotics requires the precise placement and subsequent operation of specific nanomechanical devices at particular locations. The structural programmability of DNA makes it a particularly attractive system for nanorobotics. We have developed a cassette that enables the placement of a robust, sequence-dependent DNA robot arm within a two-dimensional (2D) crystalline DNA array. The cassette contains the device, an attachment site, and a reporter of state. We used atomic force microscopy to demonstrate that the rotary device is fully functional after insertion. Thus, a nanomechanical device can operate within a fixed frame of reference.

Branched DNA has proved to be a very useful and exciting medium for nanotechnology(1). This is a consequenceof theprogrammabilityofDNAtopologyandthreedimensional (3D) structure through sequence, combinedwiththewell-defined localstructureof intermolecularassociationthat occursvia sticky- ended cohesion (2). The development of stiff motifs (3) hasenabledthe self-assemblyof DNA componentsto produce2D arraysof highquality at atomic force microscopy (AFM) resolutions (4). In a separate but related thread, robust, sequence-dependent DNA nanomechanical devices have also been developed. The insertion of such nanomechanical devices into 2D arrays results in a nanorobotic system, wherein nanoscale moving parts can be controlled relative to a fixedframe of reference. We report the development of a cassette that contains both a rotary deviceandthefeaturesthat enableitsinsertioninto an arrayat a specificsite.A changein the device control sequences or in the insertion sequences would result,respectively, in differentcontrolling elements or in a different site of insertion, all withinthe contextof the same cassette motif.

The PX-JX2 device is a robust, sequencedependentDNAmachinewhosestateiscontrolled by hybridization topology (5). It can assume two structural states [termed PX (paranemic crossover) and JX2 (paranemic crossover with two juxtaposedsites)]that differ from each other by a half-turn rotation of one end of the molecule relative to the other end. Two different pairs of set strands can bind to the framework

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

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

Fig. 1. (A) A viewperpendicular tothe plane of thecassettein the PX state.The PXstate issetbythegreenstrands inthemiddleof theupper twodomains.The reporterhairpinis seen end-onprotruding from the plane. The sticky ends on the bottom domainattach the cassette to the 2Darray.(B) Thesamemoleculeis shownobliquelyso the reporterhairpin can be seen. (C) A view similarto (A), exceptthatthe cassette is in theJX2 state,which is setbythepurple strands.The reporterhairpin is now behind the cassette, a point emphasized in (D). All drawingsareinavirtual-bondrepresentationproducedbytheprogramGIDEON

(13). (E) A 5% polyacrylamide gel run in TAEMg buffer (3). The two different states are shown both for a cassette without a hairpin and for a cassette includinga reporterhairpin.The single bands in each lane indicatethat the motifsare stableand monodisperse.BP, base pair.

w.sciencemag.org SCIENCE VOL 314 8 DECEMBER 2006 1583 on July 24, 2009 w.sciencemag.org Downloaded from of the device, thereby establishing which structural state it adopts. Different devices can be addressedindependently, leadingto 2N structural statesif N devicesare present(6). We showedby

AFM that the PX-JX2 device is functionalafter the cassette has been inserted into a 2D DNA array. Thec assetteu sedh erea lsoc ontains ac om- ponent that reports its state, although that is not a general requirement.

Thecassettethatwe havedevelopedis shown schematicallyin bothof its statesin Fig.1. A and B show the cassetteplus a reporterhairpin in the PX state:A is perpendicularto its plane,and B is oblique;C and D show similarviewsof the JX2 state. The cassette consists of three helical domains, one of which is much shorter than the other two. The short domain, shown on the bottomin Fig. 1, A to D, is the insertion domain. It contains sticky ends that enable its cohesion roughly perpendicular (three nucleotide pairs rotation,~103°)to the arraythat supportsit. This

Fig. 2. The arrays are shown schematically to demonstrate the two states of the device in the cassette. The eight TX tiles that form the array are shown in differently colored outlined tiles. For clarity, the cohesive ends are shown to be the same geometrical shape, although they all contain different sequences. The cassette and reporter helix are shown as solid red components; the marker tile is labeled M and is shown with a solid black rectangle representing the domain of the tile that protrudes from the rest of the array. Both the cassette and the marker tile are rotated ~103° from the other components of the array (three nucleotides rotation). The PX arrangement is shown on the left, and the JX2 arrangement is on the right. The reporter hairpin points toward the marker tile in the PX state but points away from it in the JX2 state.

Fig. 3. (A) Conversion of the array in the PX state to the array in the JX2 state. (B) The reverse motion, JX2 to PX. The scales of the AFM images are indicated by a 100-nm scale bar in the upper right of each image. In both states, the cassette and the marker tile are visible as a doubly lobed blob-like region. In the PX state, the reporter hairpin is visible as a bright spot at the center of the blob. In the JX2 state, the reporter arm is visible as a bright spot on one

edge of the blob. We have emphasized these features on the four images: In each image, we have drawn a black box around the unit cell repeat in two cases and a blue rounded figure encircling the blob-like region. In one of the two boxes, we have emphasized the reporter arm by enclosing it in a red circle. Expanded, upright double-scale copies of these boxes are shown adjoining the upper left edge of each image. In addition, in the lower left corner of each image, we have taken a 50 by 50-nm portion of the image, circled the marker in a black ellipse, and enclosed the cassette with a red curve that has a protrusion corresponding to the reporter hairpin. The right side of each pair of images is from an aliquot taken from a solution of the material on the left and then converted to the other state.

8 DECEMBER 2006 VOL 314 SCIENCE w.sciencemag.org1584 on July 24, 2009 w.sciencemag.org Downloaded from domainis connectedto the middle domainby a DAO (double crossover with antiparallel helices whose crossoversareseparatedbyanoddnumber of DNA half-turns) linkage (3). In contrast,the central domainis connectedto the upper domain by a PX linkage (7). Further right on the upper domains, the double-helical continuity is interrupted by a pair of set strands (green in A and B, purple in C and D) that controlsthe state of the

PX-JX2 device. Proceeding to the right, the twohelixmotif continues forabout four double-helical turns.A long reporterhairpin has been attachedso that it extends perpendicular to the plane of the cassette.This hairpinpointsin opposite directions in the PX state and in the JX2 state, enabling differentiationof thetwostatesby meansofAFM.

Thestabilityofthecassetteinbothstates,withand without the reporter hairpin, is indicatedby the presenceofsinglebandsona nondenaturinggel in Fig.1E.

A three-domaintile (TX) array(8) was selected forinsertion.In thisarray, theTX tilesareconnected sothatthebottomdomain ofeach tileis attachedto the upperdomainof a tile in an adjacentcolumn (Fig.2). Thisarrangementproducesslotsthat may be flankedby sticky ends on the terminiof the middledomainsof eachTX tile.Thesestickyends canbeusedtobindanothertilewithcomplementary stickyendsin thatsite (8). We formthe TX array with eightuniquetiles,so as to accommodatethe cassette's long reporter hairpin(Fig.1); the size of the hairpin needed to demonstrate motion has limitedus to only two insertedelements.One of theseelementsisthecassette,containingthePX-JX2 device,andthe otheris a TXmarkertile,parallelto the cassette,thatenables us to establisha reference frameon the array. The markertileis in the same columnas the cassette insertion domain (Fig. 2). Thesequencesofthecassetteandthetilesareshown infig.S1;the presenceof allstrandsin eachstateis shownin figs.S2and S3;theconversionofstatein solution is shown in fig. S4 (9).

The results of insertionand state conversion are shownby AFM in Fig. 3. Fig. 3A shows an array of PX-state cassettes (left) that have been converted to JX2-state cassettes(right); Fig. 3B shows the reverse conversion, where an array formed with JX2-statecassettes(left)is converted to cassettes in the PX state(right).It is important to recognizethattheseconversionsoccur after the cassettes have been inserted into the array [detailedmethods are describedin (9)].In addition to the arrays shown in Fig. 3, we have examined two other sets of insertedcassette arrays(figs.S5 and S6) (9). As summarized in table S1 (9), the AFM images are only good enough to ascertain the states of about half of the pretransition cassettesand slightly fewer of the posttransition cassettes. Amongthethreeimage sets(Figs.3 and figs. S5 and S6), we detected no errors in the pretransition arrays. After conversionfromthe PX state,95 of 96 cassettesare seen correctlyin the

JX2 state; after conversionfrom the JX2 state,85 of 86 cassettesare seen correctlyin the PX state, suggestinga conversionerror rate ~1%.

It is crucialfor nanorobotics to be able to insert controllable devices into a substrate, thereby leading to a diversity of structural states. Here we have demonstratedthat a single device has been inserted and converted at a specific site. There is no reason to expectthatthe systemis limited to a single device unit; as noted above, the specific addressability of the two-statePX-JX2 device has been demonstrated previously (6). It has been pointed out that two opposing PX-JX2 devices couldb e usedt op roducec omplex patterns (10).

The eight-tileTX array used here is technically difficult to obtain, but the recent advance in simplified 2D DNA patterning by Rothemund (1) should facilitatethe constructionof complex baseplanesforthesesystems.Similarly,DNAtubes (12) provide a means to incorporate nanomechanical devices intononplanar 2D arrangements.

References and Notes 1. N. C. Seeman, P. S. Lukeman, Rep. Prog. Phys. 68, 237 (2005). 2. H. Qiu, J. C. Dewan, N. C. Seeman, J. Mol. Biol. 267, 881 (1997). 3. T.-J. Fu, N. C. Seeman, Biochemistry. 32, 3211 (1993). 4. E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature 394, 539 (1998). 5. H. Yan, X. Zhang, Z. Shen, N. C. Seeman, Nature 415,6 2 (2002). 6. S. Liao, N. C. Seeman, Science 306, 2072 (2004). 7. Z. Shen, H. Yan, T. Wang, N. C. Seeman, J. Am. Chem.

Soc. 126, 1666 (2004). 8. T. LaBean et al., J. Am. Chem. Soc. 122, 1848 (2000). 9. Materials and methods, additional AFM images, and gel evidence for cassette formation are available as supporting material on Science Online. 10. A. Carbone, N. C. Seeman, Proc. Natl. Acad. Sci. U.S.A. 9, 12577 (2002). 1. P. W. K. Rothemund, Nature 440, 297 (2006). 12. W. B. Sherman, N. C. Seeman, Biophys. J. 90, 4546 (2006). 13. J. J. Birac, W. B. Sherman, J. Kopatsch, P. E.

Constantinou, N. C. Seeman, J. Mol. Graph. Model. 25, 470 (2006). 14. We thank R. Sha for valuable discussions and C. Mao,

H. Yan, and N. Jonoska for useful comments on the manuscript. This research has been supported by grants from the National Institute of General Medical Sciences, NSF, the Army Research Office, and Nanoscience Technologies. N.C.S. is a scientific advisor to Nanoscience

Technologies. Both the PX-JX2 device and 2D DNA arrays have had patent applications submitted. The 2D arrays are U.S. patent 6,255,469.

Supporting Online Material w.sciencemag.org/cgi/content/full/314/5805/1583/DC1 Materials and Methods SOM Text Figs. S1 to S6 Table S1 References

Enzyme-Free Nucleic Acid

Logic Circuits Georg Seelig,1 David Soloveichik,2 David Yu Zhang,2 Erik Winfree2,3*

Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use singlestranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.

To date, no man-made chemical circuits even remotely approach the complexity and reliability of silicon-based electronics. Once reliable principles for their design are established, syntheticchemicalcircuitscould be used routinely to control nanoscale devices in vitro, to analyze complex chemical samples in situ, or to interface with existing biological cir- cuits in vivo (1). Construction of synthetic biological circuits de novo is a powerful test of design principles (2).

Rational design of nucleic acid devices is simplified by the predictability of Watson- Crick base pairing; thus, nucleic acids are a promising alternative to proteins for synthetic chemical circuits. Allosteric ribozymes that take small molecules as input have been shown to perform logical functions (3). However, their output (a cleaved or ligated oligonucleotide) is of a different form than the input; hence, cascading is difficult. Automata performing multiple logical operations in parallel (4), single-step signaling cascades (5),

1Department of Applied Physics, California Institute of Technology, Pasadena, CA 91125, USA. 2Departmentof Computation and Neural Systems, California Institute of Technology, Pasadena, CA 91125, USA. 3Department of Computer Science, CaliforniaInstitute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

*To whom correspondence should be addressed. E-mail: winfree@caltech.edu w.sciencemag.org SCIENCE VOL 314 8 DECEMBER 2006 1585 on July 24, 2009 w.sciencemag.org Downloaded from

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