Biomacromolecules 2005, 6, 2528-2532
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A New Triple Crossover Triangle (TXT) Motif for DNA Self-Assembly Bryan Wei and Yongli Mi* Department of Chemical Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Received March 30, 2005; Revised Manuscript Received June 9, 2005
A new triple crossover triangle (TXT) motif was conceived on the basis of the DNA triple crossover (TX) motif. The new motif is rigid and triangular prism-shaped, out of which 1-D, 2-D, and 3-D DNA structures can be assembled. The 1-D TXT array was self-assembled and was observed by the TEM with negative staining by uranyl acetate. The architectural schemes for 2-D and 3-D structures are presented. Introduction The powerful molecular recognition system of DNA base pairing can be used in nanotechnology to direct selfassemblies of DNA architecture. DNA self-assembly has been found to be an innovative methodology for preparing nanoscaled patterns.1 DNA architecture involves the following: first, selecting sequences of DNA oligonucleotides for creating specific topologies, shapes, and arrangements of secondary and tertiary structures; second, enabling them to self-assemble into a desired structure; and third, modifying the DNA templates or scaffolds for specific applications.2 In DNA nanotechnology, a variety of rigid DNA motifs have been obtained, such as the DNA double crossover (DX),3 the DNA triple crossover (TX),4 and the DNA paranemic crossover (PX),5-7 from which, 1-D and 2-D arrays can be attained.3,4,8-11 In particular, some 3-D structures can also be constructed by careful designs of the branched DNA motifs, including the famous DNA cube12 and the octahedron.13 In our report, we present a new DNA motif, the triple crossover triangle (TXT), which is a motif derived from the original TX motif. It consists of eight oligonucleotides hybridized to form three double-stranded helices in the shape of triangular prism and linked by strand exchanges at six immobile crossover points. It has six sticky ends, three on each side of the TXT motif, with which the TXT motif can be joined together in 1-D, 2-D, and 3-D arrays by the desired sticky ends matching strategy. There are at least three reasons why we introduce the TXT motif to DNA self-assembly research. First, because the motif is in a triangular prism shape, it is almost certainly more rigid than the existing planar DNA motifs. Second, in the 2-D array, the TXT motif can be self-assembled following the algorithm of the DX array,8 and, in addition, leaving an extra discontinuous DNA double helices layer above, which is open for further modifications without affecting the desired array. Because of the extra layer of the DNA double helices, the height of the array is doubled. Third, most importantly, * Corresponding author. E-mail:
[email protected].
the TXT motif can be used for building 3-D arrays. With appropriate sticky ends matching strategies, TXT motifs, which are by themselves 3-D, can be packed to form 3-D arrays. In our experiment, we have obtained the selfassembled TXT 1-D array, which was visualized by transmission electron microscopy (TEM). The designs of 2-D and 3-D arrays are presented. Another independent work, also dealing with the similar TXT DNA motif, published in Nanoletters (Nanolett. 2005, 5, 693-696), was brought to our attention during the review process of this work. Materials and Experimental Section Design of the Complexes. The sequence and the superstructure of our triple crossover triangle (TXT) motif were modified from the sequence of the triple crossover (TX) motif with the program of SEQUIN (S1) to minimize the chance of undesired complementarity and sequence symmetry.11 The original TX motif consists of seven oligonucleotides, hybridized to form three double-stranded helices lying in a plane and linked by strand exchange at four immobile crossover points. The derived TXT motif, in our research, consists of eight oligonucleotides, hybridized to form three doublestranded helices in a triangular prism geometry, rather than lying in a plane, by strand exchange at six immobile crossover points as shown in Figure 1. The sequences of the eight oligonucleotides for the TXT motif are also shown in Figure 1. The dashed and solid lines between the DNA double helices represent the crossovers. As seen from Figure 1A, there are only three or four base pairs between two crossover points in certain locations, which may cause the stability concern about the motif. However, even if the base pairings in those locations are not stable, the overall stability of the TXT structure does not rely on them only. Instead, the overall stability contribution is from all the complementary base pairs. Synthesis and Purification of DNA. Custom oligonucleotides were purchased from Invitrogen Corp. (www.invitrogen.com). DNA strands were purified by electrophoresis;
10.1021/bm050230b CCC: $30.25 © 2005 American Chemical Society Published on Web 07/21/2005
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TXT Motif for DNA Self-Assembly
Figure 1. A represents the line drawing of the TXT motif, consisting of eight oligonucleotides, hybridized to form three double-stranded helices in a triangular prism geometry by strand exchange at six immobile crossover points. The dashed and solid lines between the DNA double helices represent the crossovers. B shows the section view of the TXT motif. The three green circles stand for three DNA helices. In each helix, the angle between adjacent crossover points is around 120°.
Figure 2. The schematic demonstration of the 1-D array of the TXT motif. The three sticky ends A, B, and C of one TXT motif are matched with the counter three sticky ends a, b, and c of another TXT motif. The coupling regime is A to a, B to b, and C to c. Table 1. Matching Scheme of TXT Motifs array 1-D 2-D cf. Figure 4 3-D cf. Figure 5
complementary sticky ends ATa ATd
BTb BTc
CTc
bTC A 1 T a3
aTD A2 T a 1
A3 T b 2
B 1 T b3 a′3 T A′1 b′3 T B′1
B2 T b 1 a′1 T A′2 b′1 T B′2
B3 T a 2 b′2 T A′3 a′2 T B′3
bands were tailored out of 15% denaturing polyacrylamide gels and eluted in a solution containing 500 mM ammonium acetate, 10 mM magnesium acetate, and 1 mM EDTA. Formation of Hydrogen-Bonded Complexes. Complexes were formed by mixing a stoichiometric quantity of each strand estimated by OD260 in 20 mM Tris (pH 7.6), 2 mM EDTA, and 12.5 mM MgCl2 (TAE/Mg). The final concentration of each DNA strand was 1.0 µM, and the final volume was 50 µL. Oligonucleotide mixtures were cooled slowly from 95 to 4 °C in a heating block for 51 h to facilitate hybridization. TEM Imaging. Images of were obtained on a JEOL JEM 2010 TEM. Direct observation of DNA structures was carried out by pipetting 10 µL of annealed DNA solution onto a copper grid with porous carbon film. The copper grid was
Figure 3. The TEM images of the 1-D array of the TXT motif. The scale bars are 200 nm.
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Wei and Mi
Figure 4. A is the schematic demonstration of the 2-D array of the TXT motif. B and C are the single TXT motifs. D and E show the crosssections of the 2-D array along the cutting planes of section I and section II in A. F is a bird’s-eye view of the 2-D array of the top layer only, and the bottom layer is omitted. The sticky ends matching strategy is given in Table 1.
placed on a piece of filter paper, so that the buffer solution could be removed by the filter paper. The sample grid was then moved to a silicon wafer. Negative staining was adopted by first pipetting 10 µL of 2% uranyl acetate solution onto the sample grid for 5 min. The excess liquid was then removed from the sample by lightly touching an edge of filter paper to the edge of the grid. The sample grid was put in the desiccator overnight before the TEM examination.
Results and Discussions 1-D Array. The designed sticky ends matching strategy enables the self-assembly of the TXT motifs to occur in one dimension only. The schematic diagram of the 1-D array is shown in Figure 2. The three sticky ends A, B, and C of one TXT motif are matched with the counter three sticky ends a, b, and c of another TXT motif, leading to the 1-D array. The coupling regime is A to a, B to b, and C to c, as given in Table 1. The TEM images of the 1-D array of the TXT motif are shown in Figure 3A,B. The scale bars in
Figure 3 are 200 nm. The nonuniformity is due to two factors: One is from the overlapping effect or bundling effect of the TXT 1-D array. The other is from the enlargement effect of sample staining. We suggest that the TXT motif, in which the three double helices of DNA joining together to form a triangular prism geometry are expected to be more rigid than the existing PX, DX, and TX motifs, in which two or three double helices of DNA joined together in a planar pattern.3,4,7 Furthermore, by the designed sticky ends matching strategies, 2-D and 3-D arrays can also be obtained. 2-D Array. The assembled 2-D array is shown schematically in Figure 4A. The single TXT motifs, the basic structural units, are shown in Figure 4B,C. The sticky ends matching strategy is demonstrated in Table 1 as A to d, B to c, a to D, and b to C. The sticky ends are labeled in Figure 4B,C, whereas the nonsticky ends of the top DNA double helices are not labeled. Figure 4D,E show the cross-sections of the 2-D array along the cutting planes of section I and section II in Figure 4A. The 2-D array has a double-layered
TXT Motif for DNA Self-Assembly
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Figure 5. A is the schematic demonstration of the 3-D array of the TXT motif. B, C, D, and E are the single TXT motifs. The sticky ends matching strategy is demonstrated in Table 1 according to the sticky ends as labeled in B, C, D, and E. A specified site of 3-D assembly is given by the highlighted motifs in F, in which a TXT motif (green) associates with other six TXT motifs (three on each side). The TXT motif (green) in F (I or II) joins with two red and one blue TXT motifs on the one end in F (I) and on the other end joins with another two red and one blue TXT motifs accordingly in F (II).
structure. The bottom layer is a continuously arrayed plane as shown in Figure 4D,E by the solid circles, whereas for the top layer, the dashed circles are one motif behind the solid circle. The top layer is discontinuous as shown in Figure 4F, which is a bird’s-eye view of the top layer only, while the bottom layer is omitted. The discontinuous DNA double helices of the top layer are open for further modifications. 3-D Array. The assembled 3-D array is shown schematically in Figure 5A. The single TXT motifs, the basic structural units, are shown in Figure 5B-E. The sticky ends matching strategy is demonstrated in Table 1 according to the sticky ends as labeled in Figure 5B-E. Figure 5F further illustrates the sticky end matching strategy at section I and section II of Figure 5A. A specified site of 3-D assembly is given by the highlighted motifs in Figure 5F, in which a TXT motif (green) associates with the other six TXT motifs (three on each side). The TXT motif (green) in Figure 5F (I or II) joins with two red and one blue TXT motifs on the one end
in Figure 5F (I) and on the other end, joins with another two red and one blue TXT motifs accordingly in Figure 5F (II). Acknowledgment. The financial support of the RGC research grant, 602603, is greatly acknowledged. References and Notes (1) Seeman, N. C. Nature (London) 2003, 421, 427-431. (2) Seeman, N. C. Trends Biotechnol. 1999, 17, 437-442. (3) Li, X.; Yang, X.; Qi, J.; Seeman, N. C. J. Am. Chem. Soc. 1996, 118, 6131-6140. (4) 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. (5) Seeman, N. C. Nano Lett. 2001, 1, 22-26. (6) Zhang, X.; Yan, H.; Shen, Z.; Seeman, N. C. J. Am. Chem. Soc. 2002, 124, 12940-12941. (7) Shen, Z.; Yan, H.; Wang, T.; Seeman, N. C. J. Am. Chem. Soc. 2004, 126, 1666-1674. (8) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature (London) 1998, 394, 539-544.
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(9) Liu, F.; Sha, R.; Seeman, N. C. J. Am. Chem. Soc. 1999, 121, 917922. (10) Liu, D.; Park, S. H.; Reif, J. H.; LaBean, T. H. PNAS 2004, 101, 717-722. (11) Li, H.; Park, S. H.; Reif, J. H.; LaBean, T. H.; Yan, H. J. Am. Chem. Soc. 2004, 126, 418-419.
Wei and Mi (12) Chen, J.; Seeman, N. C. Nature (London) 1991, 350, 631633. (13) Shih, W. M.; Quispe, J. D.; Joyce, G. F. Nature (London) 2004, 427, 618-621.
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