Two-Dimensional (2D) DNA Crystals Assembled from Two DNAStrands

Haipeng Liu, Yu He, Alexander E. Ribbe, and Chengde Mao*

Purdue University, Department of Chemistry, 560 Oval Drive, West Lafayette, Indiana 47907

Received August 31, 2005; Revised Manuscript Received September 21, 2005

Sequence symmetry has been used to simplify the design of a DNA double-crossover (DX) molecule. Theresulting DX molecule can self-assemble into two-dimensional (2D) crystalline arrays, but only requirestwo instead of otherwise four different DNA strands.

This paper reports a hierarchical DNA self-assembly. Twodifferent DNA single strands first assemble into four-strandedDNA double-crossover (DX) structures, which then assembleinto two-dimensional (2D) crystalline arrays. The key to thiswork is application of DNA sequence symmetry. Sequencesymmetry has been intentionally avoided in previous designsof DNA nanostructures in order to minimize unwantedsecondary structures.1

DNA self-assembly is an effective way to produce patternsat the nanometer scale.2 Specially designed single DNAstrands can self-assemble into well-defined motifs, and then,the motifs self-assemble into final structures, includingindividual geometric or topologic structures, 1D or 2Dperiodic or aperiodic arrays.3 The quality of the finalstructures critically depends on the intermediate motifs.However, the current available DNA motifs are quitecomplicated and normally consist of several different DNAstrands at particular ratios. Though we could, in principle,adjust the ratios of DNA strands to any value as desired, itis experimentally difficult. DNA concentration is commonlyestimated by optical absorbance at 260 nm, which is themaximum absorption wavelength of DNA. This estimationis known to be inaccurate because of different compositionsand secondary structures of DNA molecules. Pairwisetitration of DNA strands on native polyacrylamide gelelectrophoresis (PAGE) can improve the ratio accuracy,4 butthis method is limited by the detection sensitivity. To thebest, we may reliably adjust the ratio to 95% accuracy inexperiment. If multiple DNA strands are involved, theproblem becomes even worse. One effective approach tosolve this problem is to reduce the number of different DNAstrands. If only a very few DNA strands are used, it wouldbe much easier to adjust the ratios. With the accompanyingreduction of the number of DNA strands, the unique DNAsequence space decreases, which simplifies DNA sequencedesign. This advantage will facilitate the design of compli-cated nanostructures. Recently, we have successfully appliedDNA sequence symmetry to simplify a cross-DNA motifand design a 3-point-star motif.5 Here, we extend this strategyeven more vigorously to simplify the design of a four-stranded DX motif (Figure 1). Formerly, a DX motif consists

of four different DNA strands.6 With sequence symmetry, aDX motif contains only two different DNA strands, whichwould be the DNA motif that requires the least differentDNA strands so far.* E-mail: mao@purdue.edu.

Figure 1. Symmetric DNA double-crossover (sDX) molecules. (a)An sDX molecule with blunt ends. The black arrows indicate a twofoldrotational axis of the sDX molecule. (b) An sDX molecule with stickyends can self-assemble into 2D crystals. (c) DNA sequences of theblunted and sticky-ended sDX molecules.

2943Biomacromolecules 2005,6, 2943-2945

10.1021/bm050632j CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 10/07/2005

Figure 1 illustrates the molecular design of symmetricDNA double-crossover (sDX) molecules. A DX moleculeis composed of two pseudo-helices. These DNA helices layside by side and are joined together by strand crossoversbetween them at two positions (crossover points). One blunt-ended sDX molecule consists of four strands (Figure 1a):two identical red strands and two identical blue strands. Atwofold rotational axis runs through the molecule, as shownby two arrowheads. The DNA sequences between the twocrossover points that result from this twofold symmetry arepalindromes. The full DNA sequences are shown in Figure1c. When containing single-stranded overhangs (sticky ends),sDX molecules can associate with each other and arrangethemselves into 2D crystalline arrays (Figure 1b).

The sDX molecules are readily formed and are stableunder native conditions. After purification with denaturingpolyacrylamide gel electrophoresis (PAGE), mixture solu-tions containing DNA strands at designated ratios are slowlycooled from 95°C to room temperature in 2 h and thenanalyzed by native PAGE (see Supporting Information forexperimental details). All DNA complexes migrate as sharpbands with expected mobilities, indicating that the DNA

complexes form and are stable (Figure 2). At high enoughDNA concentration, strand1 might form oligomers otherthan dimers. However, no oligomers other than dimers havebeen observed in the gel where the DNA concentrationis ∼5 µM, which is significantly higher than the com-monly used DNA concentration (0.01-1 µM) for DNA 2Dassembly.

We characterized the self-assembly behavior of the sDXwith atomic force microscopy (AFM, Figure 3), followingestablished methods.3a DNA assemblies appeared very long(up to 30µm), but quite narrow (less than 1µm). Whenscanned at small scales (about 2µm), DNA assembliesclearly showed regular patterns at the nanometer scale. Theapparent structures were very similar to previously reportedDNA 2D arrays assembled from traditional DX molecules.3a

There were periodic band structures along the long axes ofthe 2D arrays. The observed repeating distance was 14 nm,roughly equal to the calculated length (52 bp× 0.33 nm/bp) 13.86 nm) of an sDX model.

We have also observed periodic patterns perpendicularto the long axes of the DNA 2D arrays, with repeatingdistance of 7 nm. This phenomenon can also be found inprevious reports.7,8 We believe that it is due to electrore-pulsion between the negatively charged DNA backbones.Between the two crossovers in a DX molecule, the compo-nent DNA duplexes are short (16 base pairs) and are forcedto be close to each othersalmost parallel to each other.Beyond the crossover points, constraints between the DNAduplexes become much weaker. The DNA duplexes willslightly move away from each other to minimize therepulsion between the like charges on DNA duplexes. Thus,the DNA duplexes are not parallel to each other anymore. This modified DX model can fit the AFM data nicely(Figure 4).8

In conclusion, we have applied sequence symmetry tominimize the strand usage in the formation of DNA nano-structures. The symmetric motif not only reduces the numberof DNA strands needed but also greatly eases experimentaldifficulty, an important but often underestimated issue.Furthermore, this method decreases the unique sequence

Figure 2. Characterization of sDX molecules by native polyacryl-amide gel electrophoresis (PAGE, 10%). The left-most lane containsthree single DNA strands as size markers, which are 26, 53, and100 nucleotides (nt) long, respectively.

Figure 3. Atomic force microscopy (AFM) analysis of DNA 2D crystals assembled from sticky-ended sDX molecules. From left to right, theimages are from three different scanning sizes.

2944 Biomacromolecules, Vol. 6, No. 6, 2005 Communications

space. This feature might be greatly appreciated in a com-plicated system where a large number of unique DNA se-quences are needed. More fundamentally, we would like toask, what is the minimum number of DNA strands requiredfor the formation of well-defined DNA nanostructures? Thisis a basic question for understanding the capability and the

limitations of structural DNA nanotechnology. The currentreport is an effort along this direction.

Acknowledgment. This work was supported by NSF(EIA-0323452), DARPA/DSO (MDA 972-03-1-0020), andPurdue University (a startup fund). AFM study was carriedout in the Purdue Laboratory for Chemical Nanotechnology(PLCN).

Supporting Information Available. Experimental de-tails. This material is available free of charge via the Internetat http://pubs.acs.org.

References and Notes

(1) Seeman, N. C.J. Biomol. Struct. Dyn.1990, 8, 573-581.(2) Seeman, N. C.Nature (London)2003, 421, 427-431.(3) (a) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C.Nature

(London)1998, 394, 539-544. (b) LaBean, T. H.; Yan, H.; Kopatsch,J.; Liu, F.; Winfree, E.; Reif, J. H.; Seeman, N. C.J. Am. Chem.Soc.2000, 122, 1848-1860. (c) Mao, C.; Sun, W.; Seeman, N. C.J. Am. Chem. Soc.1999, 121, 5437-5443. (d) Yan, H.; Park, S. H.;Finkelstein, G.; Reif, J. H.; LaBean, T. H.Science2003, 301, 1882-1884. (e) Liu, D.; Wang, M.; Deng, Z.; Walulu, R.; Mao, C.J. Am.Chem. Soc.2004, 126, 2324-2325. (f) Ding, B.; Sha, R.; Seeman,N. C. J. Am. Chem. Soc.2004, 126, 10230-10231. (g) Rothemund,P. W. K.; Papadakis, N.; Winfree, E.PLoS Biology2004, 2, 2041-2053. (h) Shih, W. M.; Quispe, J. D.; Joyce, G. F.Nature (London)2004, 427, 618-621. (i) Malo, J.; Mitchell, J. C.; Venien-Bryan, C.;Harris, J. R.; Wille, H.; Sherratt, D. J.; Turberfield, A. J.Angew.Chem. Int. Ed.2005, 44, 3057-3061. (j) Mathieu, F.; Liao, S.;Kopatsch, J.; Wang, T.; Mao, C.; Seeman, N. C.Nano Lett.2005, 4,661-665. (k) Park, S. H.; Barish, R.; Li, H.; Reif, J. H.; Finkelstein,G.; Yan, H.; LaBean, T. H.Nano Lett. 2005, 4, 693-696. (l)Chworos, A.; Severcan, I.; Koyfman, A. Y.; Weinkam, P.; Oroudjev,E.; Hansma, H. G.; Jaeger, L.Science2004, 306, 2068-2072. (m)Chelyapov, N.; Brun, Y.; Gopalkrishnan, M.; Reishus, D.; Shaw,B.; Adleman, L.J. Am. Chem. Soc.2004, 126, 13924-13925.

(4) Kallenbach, N. R.; Ma, R.-I.; Seeman, N. C.Nature (London)1983,305, 829-831.

(5) (a) He, Y.; Chen, Y.; Liu, H.; Ribbe, A. E.; Mao, C.J. Am. Chem.Soc.2005, 127, 12202-12203. (b) He, H.; Tian, Y.; Chen, Y.; Deng,Z.; Ribbe, A. E.; Mao, C.Angew. Chem. Int. Ed.Early View paper,published online Sept 27, 2005.

(6) Fu, T.-J.; Seeman, N. C.Biochemistry1993, 32, 3211-3220.(7) Rothemund, P. W. K.; Ekani-Nkodo, A.; Papadakis, N.; Kumar, A.;

Fygenson, D. K.; Winfree, E.J. Am. Chem. Soc.2004, 126, 16344-16352.

(8) Hariadi, R. F. http://www.rpgroup.caltech.edu/courses/aph161/Lecture/Poster%20RH.pdf (accessed on August 29, 2005).

BM050632J

Figure 4. A modified DX model and its fit with an experimental AFMimage.

Communications Biomacromolecules, Vol. 6, No. 6, 2005 2945