Biomacromolecules 2005, 6, 2943-2945
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Two-Dimensional (2D) DNA Crystals Assembled from Two DNA Strands 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. The resulting DX molecule can self-assemble into two-dimensional (2D) crystalline arrays, but only requires two instead of otherwise four different DNA strands. This paper reports a hierarchical DNA self-assembly. Two different DNA single strands first assemble into four-stranded DNA double-crossover (DX) structures, which then assemble into two-dimensional (2D) crystalline arrays. The key to this work is application of DNA sequence symmetry. Sequence symmetry has been intentionally avoided in previous designs of DNA nanostructures in order to minimize unwanted secondary structures.1 DNA self-assembly is an effective way to produce patterns at the nanometer scale.2 Specially designed single DNA strands can self-assemble into well-defined motifs, and then, the motifs self-assemble into final structures, including individual geometric or topologic structures, 1D or 2D periodic or aperiodic arrays.3 The quality of the final structures critically depends on the intermediate motifs. However, the current available DNA motifs are quite complicated and normally consist of several different DNA strands at particular ratios. Though we could, in principle, adjust the ratios of DNA strands to any value as desired, it is experimentally difficult. DNA concentration is commonly estimated by optical absorbance at 260 nm, which is the maximum absorption wavelength of DNA. This estimation is known to be inaccurate because of different compositions and secondary structures of DNA molecules. Pairwise titration of DNA strands on native polyacrylamide gel electrophoresis (PAGE) can improve the ratio accuracy,4 but this method is limited by the detection sensitivity. To the best, we may reliably adjust the ratio to 95% accuracy in experiment. If multiple DNA strands are involved, the problem becomes even worse. One effective approach to solve this problem is to reduce the number of different DNA strands. If only a very few DNA strands are used, it would be much easier to adjust the ratios. With the accompanying reduction of the number of DNA strands, the unique DNA sequence space decreases, which simplifies DNA sequence design. This advantage will facilitate the design of complicated nanostructures. Recently, we have successfully applied DNA sequence symmetry to simplify a cross-DNA motif and design a 3-point-star motif.5 Here, we extend this strategy even more vigorously to simplify the design of a fourstranded DX motif (Figure 1). Formerly, a DX motif consists * E-mail:
[email protected].
Figure 1. Symmetric DNA double-crossover (sDX) molecules. (a) An sDX molecule with blunt ends. The black arrows indicate a twofold rotational axis of the sDX molecule. (b) An sDX molecule with sticky ends can self-assemble into 2D crystals. (c) DNA sequences of the blunted and sticky-ended sDX molecules.
of four different DNA strands.6 With sequence symmetry, a DX motif contains only two different DNA strands, which would be the DNA motif that requires the least different DNA strands so far.
10.1021/bm050632j CCC: $30.25 © 2005 American Chemical Society Published on Web 10/07/2005
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Figure 2. Characterization of sDX molecules by native polyacrylamide gel electrophoresis (PAGE, 10%). The left-most lane contains three single DNA strands as size markers, which are 26, 53, and 100 nucleotides (nt) long, respectively.
Figure 1 illustrates the molecular design of symmetric DNA double-crossover (sDX) molecules. A DX molecule is composed of two pseudo-helices. These DNA helices lay side by side and are joined together by strand crossovers between them at two positions (crossover points). One bluntended sDX molecule consists of four strands (Figure 1a): two identical red strands and two identical blue strands. A twofold rotational axis runs through the molecule, as shown by two arrowheads. The DNA sequences between the two crossover points that result from this twofold symmetry are palindromes. The full DNA sequences are shown in Figure 1c. When containing single-stranded overhangs (sticky ends), sDX molecules can associate with each other and arrange themselves into 2D crystalline arrays (Figure 1b). The sDX molecules are readily formed and are stable under native conditions. After purification with denaturing polyacrylamide gel electrophoresis (PAGE), mixture solutions containing DNA strands at designated ratios are slowly cooled from 95 °C to room temperature in 2 h and then analyzed by native PAGE (see Supporting Information for experimental details). All DNA complexes migrate as sharp bands with expected mobilities, indicating that the DNA
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complexes form and are stable (Figure 2). At high enough DNA concentration, strand 1 might form oligomers other than dimers. However, no oligomers other than dimers have been observed in the gel where the DNA concentration is ∼5 µM, which is significantly higher than the commonly used DNA concentration (0.01-1 µM) for DNA 2D assembly. We characterized the self-assembly behavior of the sDX with atomic force microscopy (AFM, Figure 3), following established methods.3a DNA assemblies appeared very long (up to 30 µm), but quite narrow (less than 1 µm). When scanned at small scales (about 2 µm), DNA assemblies clearly showed regular patterns at the nanometer scale. The apparent structures were very similar to previously reported DNA 2D arrays assembled from traditional DX molecules.3a There were periodic band structures along the long axes of the 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 perpendicular to the long axes of the DNA 2D arrays, with repeating distance of 7 nm. This phenomenon can also be found in previous reports.7,8 We believe that it is due to electrorepulsion between the negatively charged DNA backbones. Between the two crossovers in a DX molecule, the component DNA duplexes are short (16 base pairs) and are forced to be close to each othersalmost parallel to each other. Beyond the crossover points, constraints between the DNA duplexes become much weaker. The DNA duplexes will slightly move away from each other to minimize the repulsion between the like charges on DNA duplexes. Thus, the DNA duplexes are not parallel to each other any more. This modified DX model can fit the AFM data nicely (Figure 4).8 In conclusion, we have applied sequence symmetry to minimize the strand usage in the formation of DNA nanostructures. The symmetric motif not only reduces the number of DNA strands needed but also greatly eases experimental difficulty, an important but often underestimated issue. Furthermore, this method decreases the unique sequence
Figure 3. Atomic force microscopy (AFM) analysis of DNA 2D crystals assembled from sticky-ended sDX molecules. From left to right, the images are from three different scanning sizes.
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limitations of structural DNA nanotechnology. The current report is an effort along this direction. Acknowledgment. This work was supported by NSF (EIA-0323452), DARPA/DSO (MDA 972-03-1-0020), and Purdue University (a startup fund). AFM study was carried out in the Purdue Laboratory for Chemical Nanotechnology (PLCN). Supporting Information Available. Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes
Figure 4. A modified DX model and its fit with an experimental AFM image.
space. This feature might be greatly appreciated in a complicated system where a large number of unique DNA sequences are needed. More fundamentally, we would like to ask, what is the minimum number of DNA strands required for the formation of well-defined DNA nanostructures? This is a basic question for understanding the capability and the
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