Letter pubs.acs.org/NanoLett
Self-Assembly of DNA Rings from Scaffold-Free DNA Tiles Yang Yang,† Zhao Zhao,†,‡ Fei Zhang,†,‡ Jeanette Nangreave,† Yan Liu,*,†,‡ and Hao Yan*,†,‡ †
Center for Single Molecule Biophysics, the Biodesign Institute and ‡Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States S Supporting Information *
ABSTRACT: We report a scaffold-free approach in which four- and six-helix DNA bundle units, assembled from a small number of single stranded DNA oligonucleotides precisely arranged in networks of contiguous and semicrossover strands, are connected into DNA nano rings. Nearly uniform structures with well-defined diameters of 53 ± 7, 81 ± 9, 85 ± 8, and 166 ± 13 nm were achieved by introducing uniform, in-plane curvature to the repeating units. We demonstrate that precise higher order assemblies can be achieved by fine tuning the particular features of the individual building blocks.
KEYWORDS: DNA rings, scaffold free, DNA nanotechnology, DNA self-assembly
A
consecutive times along the helical axis with the LS successively joining pairs of adjacent helices by forming semicrossovers between them. Here, each CS and LS consists of 21 nucleotides (nts), and both the CS and LS follow a 7 nt + 7 nt + 7 nt binding domain pattern with three neighboring LS and CS, respectively. For B-form DNA (with 10.5 bps per turn), 7 nt interhelix phasing corresponds to a dihedral angle of 240° (the angle between any three adjacent helices) with 21 bps corresponding to two full turns. The closest distance between the crossovers along any helix that is connected to two other helices is 7 bps, ensuring the 240° dihedral angle. Thus, the six CS and six LS alternatively bind to one another to form a sixhelix tubular unit that can be linked on both ends to other units to form linear bundles (as shown in the 6HB:Linear design in Figure 1). Similarly, 4 helix bundles (4HB) were designed (4HB:Linear in Figure 1) with four CS and four LS that are 32 nts long; here, each LS has an 8 nt + 16 nt + 8 nt binding domain pattern with three neighboring CSs. The end-to-end connection of the repeating units maintains 32 bp (approximately 3 full turns) gaps between semicrossovers along the same pair of neighboring helixes and 8 bp interhelix phasing (∼270°) resulting in the formation of linear 4HB. In order to induce ring formation and join the ends of the linear bundle assemblies we broke the cross-sectional symmetry of the multihelix bundles and introduced an in-plane curvature to each building block unit. We achieved this by systematically modifying the number of bps in selected subsets of the unit motifs through the addition or deletion of 1 or 2 bps,
s an information-coding polymer, DNA has proven itself as an excellent molecular building block for the organization and assembly of precise nanoscale structures.1−8 A predominant goal in DNA nanotechnology is to achieve precise arrangements of matter in three-dimensional space and control over twist and curvature is important to achieve this goal. While higher order DNA nanostructures9−11 sometimes exhibit cumulative effects from the intrinsic curvature of the unit tiles, it is difficult to control the exact features of the end product. Researchers have begun to use targeted insertion and deletion of base pairs within rigid 3D blocks,12 and/or stacked concentric double-helical rings consisting of different numbers of turns13 to generate and control the curvature of DNA nanostructures with both methods reliant on DNA origami scaffolded assembly. Herein, we report a scaffold-free approach in which multihelical DNA bundle units are assembled from a small number (8 or 12) of single-stranded DNA oligonucleotides (ssDNAs) into nearly uniform ring structures with welldefined diameters by introducing uniform, in-plane curvature to the unit tiles. All of the designs reported here are based on repeating tubular units; the number of unique strands required to form the individual units are 2× the number of helices in the corresponding bundles (12 strands for a six-helix bundle; 8 strands for a four-helix bundle). The constituent strands fall into one of two categories: half of them are contiguous strands (CS) that are arranged linearly and the other half strands are linking strands (LS) that each form a crossover from one helix to a neighboring helix. For example, the basic unit of the sixhelix bundle (6HB:Linear) illustrated in Figure 1 is composed of six CS (black lines) and six LS (orange). Each helix in the bundle contains a unique CS, repeated N (number of units) © 2013 American Chemical Society
Received: March 7, 2013 Revised: March 25, 2013 Published: March 27, 2013 1862
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Figure 1. Right: Schematic design of 6HB and 4HB linear bundles (all gray) formed from repeating units and circular bundles (multicolor) formed from modified repeating units, respectively. The helices are labeled from A to F in 6HB and A to D in 4HB. In 6HB:Linear, each helix is 21 bps per two turns (10.5 bps per turn) and the 7-bp binding domain ensures a 240° dihedral angle between three neighboring helixes. In the 6HB: EF+BC−, the red colored helices (E and F) are 22 bps per 2 turns and the red arrowheads point to the positions where the extra nucleotides were inserted. The blue-colored helices (B and C) are 20 bps per two turns and the blue inward pointing arrow pairs mark the positions where nucleotides were deleted. In the 4HB:Linear, each helix is 32 bps per three turns (10.66 bps per turn). The 8 bp binding domains ensure 270° dihedral angles between three neighboring helices. The pink and cyan colored helices are 33 bps per three turns and 30 bps per three turns, respectively. Left: cross section and top views, respectively, of each design with accurately depicted proportions according to the experimentally measured diameters.
twisting in the 4HB.5,12,13 The length of the bundles varied from hundreds of nanometers to several micrometers (zoom out images shown in Supporting Information, Figure S1). Meanwhile, those designs with various helical length adjustments yielded ring structures of different diameters. Ring diameters and size distributions were measured from AFM images by analyzing more than 500 individual ring structures, and the histograms are presented in Figure 2. The 4HB:2+2− arrangement yielded the smallest rings with a narrow distribution around ∼53 ± 7 nm diameter, while the 6HB:1+1− produced the largest rings with a broader size distribution, 166 ± 13 nm. The ring size corresponding to each design can be predicted from both geometric and energetic considerations. Geometrically, if there are N (an integer number) units in a ring, then the ratio of the outer perimeter to the inner perimeter (Pout/Pin) will be equal to the ratio of the outer and inner diameters (Dout/Din) and the ratio of the unit lengths in the outer and inner helices (Lout/Lin) (eq 1), which can be calculated from the number of base pairs between the crossover points multiplied by the length per base pair. The difference between Din and Dout is considered to be twice the change in radius, Δr (eq 2), which is equal to the center-to-center distance between the parallel helices. Consequently, the inner diameter can be represented as a function of Δr, the unit number of base pairs (nin, nout), and the length per base pair (din, dout) (eq 3)
generating structural tension in the units that is accumulated along particular helices. For example, in the 6HB:EF+BC− structure depicted in Figure 1, the strands that comprise helices B and C were shortened to 20 bps (blue), while those in helices E and F on the opposite side of the bundle were elongated to 22 bps (red). Thus, in each structural unit of the bundle helices B and C are moderately contracted and helices E and F are extended which generates curvature directed from the midpoint of EF to the midpoint of BC. The in plane curvature in the unit is accumulated as the bundle units coassemble such that the ends of the bundle will ultimately meet and form a closed ring, ideally a circular ring with a fixed radius. Similarly, selected strands on opposite sides of the 4HB unit were adjusted by adding one or deleting two nts (total of 33 nts and 30 nts, respectively) to introduce the desired curvature (as shown in Figure 1, 4HB:CD+AB−). In both 6HB and 4HB, besides extending and contracting two pairs of helices (2+2−) on opposite sides of the bundle unit (as depicted in the cross sectional profile in Figure 1), it is also possible to selectively extend and contract a single pair of helices (1+1−) (see Figure 2 and Supporting Information, Figures S1, S5, and S7). All possible combinations of insertions and deletions were explored. Each of the designs were successfully assembled and characterized by atomic force microscopy (AFM). As shown in Figure 2 (upper panel), the unmodified designs assembled into linear bundles as expected; nearly straight 6HBs and slightly curved 4HBs were observed. The nonlinear 4HBs are likely due to the imperfect 270° dihedral angles between the helices and 3 turns based on 32 bp repeating units, which may accumulate some structural strain in the helices that cause 1863
Pout πDout L N n d = = out = out out Pin πDi n L inN n ind in
(1)
Dout − Din = 2Δr
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Figure 2. Upper panel: cross-section of the unmodified 4HB and 6HB structures and the corresponding AFM images. Lower panel: cross sections of selected designs with modified helical lengths (color legend specifying the size of the repeating units are shown in the upper right corner) and the corresponding representative zoom in AFM images. Scale bars in all AFM images are 200 nm. Statistical analyses of the measured diameter distribution of the corresponding ring structures are shown to the right of the AFM images. A single peak Gaussian function was used to fit each histogram. The average diameter (D), the standard division (σ), and the correlation coefficient (r2) are listed for each curve fit.
Table 1. Comparison of Predicted and Experimentally Observed Ring Sizes geometric prediction
energetic prediction
real size
pattern
Δr (nm)
Din‑G (nm)
N
R0
Din‑E (nm)
N
Din (nm)
4HB:2+2− 4HB:1+1− 6HB:2+2− 6HB:1+1−
2.50 2.50 × √2 2.25 × √3 2.25 × 2
50.0 70.7 77.9 90.0
16 22 37 42
18.8° 12.2° 7.7° 6.2°
60.6 95.6 100.3 123.8
19 30 47 58
53 81 85 166
Din =
2Δrn ind in nout dout − n ind in
± ± ± ±
7 9 8 13
N 16 25 40 78
± ± ± ±
2 3 4 6
both of which could deviate from the values for the undisturbed linear helical bundles. The center-to-center interhelix spacing in DNA bundles in the honeycomb lattice has been reported as 2.25 nm4,12 but is generally assumed to be 2.50 nm for helices in parallel bundles or square lattice arrangements.5,13 The length per base pair in each DNA helix bundle was measured as
(3)
Here we introduced unique structural parameters, the centerto-center distance between helices in the DNA bundle crosssection and the length per base pair in the bent DNA helices, 1864
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0.335 ± 0.007 nm.12 However, with the insertion or deletion of bps in each unit, the segments between neighboring crossover points along any pair of DNA helices are still assumed to correspond to full helical turns. For example, the distance between semicrossovers in the inner and outer helices in 4HB:2+2− are 30 and 33 bps, respectively, and the helical rise per base pair in the DNA helices is assumed to remain 0.335 nm (din = dout). With Δr expected to be 2.50 nm for the 4HB, the inner diameter of the 4HB:2+2− ring (according to eq 3) is calculated as 50 nm, which is just slightly smaller than the experimental result of 53 ± 7 nm. The geometrically predicted diameters for the 4HB:1+1− and 6HB:2+2− designs are listed in Table 1. We found that the geometrically predicted diameters are slightly smaller than the experimentally observed ones, coinciding with the left tails of the experimental distribution histograms. This indicates that there are additional forces that effect the formation of the ring structures, possibly due to resistance based on the physical properties of DNA. Meanwhile, much bigger rings were observed for the 6HB:1+1− design compared to the geometrically predicted diameter. This result is reasonable as there are four unmodified helices (gray cylinders in Figure 2) that are assumed to have a length of 21 bps × N. In order to form a ring structure the two helices closer to the outer edge are forced to stretch while those in the inner ones are forced to contract; in that way the length per base pair must deviate from an equilibrium value. The tendency to remain extended in these four helices resists the bending forces and is in opposition to ring closure. The result is the formation a much larger ring than expected. From an energetic perspective, the total energy stored within a DNA bundle structure can be expressed as a sum of stretchcompression energy (S) and bending energy (B), which was thoroughly described by Shih et al. in 2009.12 Shih developed a simple computer program to calculate the energy of a bent multiple helix bundle. Using the same parameters and assuming each of the tubular units in our design is 1/N arc of a circle with input radian values ranging from 0.1 to 180.0°, the energies were calculated and the radian of the arc per unit, R0, corresponding to the minimal energy was identified. The results are summarized in Table 1. The number of units that can be realized is described by N = 360°/R0, and inner diameters are estimated using the equation: Din = 0.335nin(N/π). Compared to the predictions based on simple geometric considerations, the energetic predictions are closer to the experimental results. For all designs except 6HB:1+1−, the predicted diameters based on minimizing the total energy in the ring are larger than the experimentally determined results. It is likely that the ring closure process is kinetically controlled, a smaller ring will form faster than a larger ring. Therefore, the experimentally measured ring sizes are generally smaller than the energetically predicted sizes. Overall, our calculations and observations indicate that the actual ring sizes are between the geometric and energetic predictions (Din‑G < Din‑real < Din‑E). The only exception is the 6HB:1+1− design that is predicted to yield a slightly smaller ring size than were observed in AFM images. In this case, it is likely that the contributions from the stretchcompression energy and the bending energy are underestimated for the pairs of unmodified helices. In addition to the previously described designs, we also examined the other possible combinations of helical unit lengths, for example A+C−, B+D−, C+A− and D+B− for the 4HB:1+1− design and all six combinations for the 6HB design.
The experimental results are presented in the Supporting Information (Table S1) and confirm both the validity of our design strategy and the limitations of the geometric and energetic prediction methods. Most of the combinations yielded similar average sizes that fall between the two predicted values as previously discussed (see Figure 3 and Supporting
Figure 3. Distribution of all combinations of the 6HB:2+2− design. All curve fits were normalized and plotted in different colors (original statistical histograms are not displayed). Inset: histogram of the mean diameter value of each combination with standard divisions as error bars. The dash lines indicate the theoretical diameters predicted geometrically (blue) and energetically (red).
Information Figure S6, the 6HB:2+2− group for an example). The observed differences between the assemblies suggest that the specific arrangement and sequences of the strands in the unit motif does influence their assembly. Occasionally, rings out of the predicted range or even spiral structures were assembled, such as 4HB:CD+AB− and 4HB:C+A− (Supporting Information Table S1, marked in red and Supporting Information, Figures S4 and S5) which might be ascribed to strand ALS- for its repeated GC fragments (-GCCGGTGGCCG- as shown in Supporting Information table of sequences). Nevertheless, the structures described here migrated as single bands in PAGE gels (Supporting Information Figure S3A), reflecting narrow size distributions regardless of any aggregation products trapped in the wells. Conveniently, the rings can be purified and recovered from either agarose gel electrophoresis, or gradient ultracentrifugation (see Supporting Information Figure S3B,C). An obvious advantage of structures formed from repeating units is the ability to efficiently capture a large number of target molecules (e.g., modified DNA probes, aptamers, proteins, or nanoparticles) by modifying very few strands in the unit motif.9,14,15 To demonstrate this we used the 6HB:FA+CD− design and replaced the CS strand on helix C (Ccs−) and/or the CS strand on helix F (Fcs+) with biotin modified strands. Biotin is a small molecule and when attached to a thick bundle through a flexible linker we do not expect it to affect the ring formation process. Ring structures with modifications on inner, outer and both surfaces were explored (Figure 4); all three designs displayed the same average ring diameters as the corresponding nonlabeled rings. After the addition of an excess of avidin (MW: 69 kDa) the presence of biotin inside and/or outside of the rings was confirmed by AFM (Figure 4). It is remarkable that, for example, each 6HB:2+2− ring contains approximately 40 × 12 = 480 strands that can be extended or modified. These structures are promising candidates to act as 1865
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Figure 4. Binding multiple targets to the rings. (a) 6HB:F*A+C*D− before the addition of avidin; * marks the helices (F and C) and continuous strands carrying biotin modifications. (b) 6HB:F*A+CD− with biotin modifications on helix F only (outer ring). After the addition of avidin a dense layer of molecules attached to the outer surface of the rings are observed. (c) 6HB:FA+C*D− with biotin modifications on helix C (inner ring). After the addition of avidin a dense layer of molecules attached to the inner surface of the ring is observed. (d) 6HB:F*A+C*D− with biotin modifications on the outer and inner rings. After the addition of avidin both the inner and outer surfaces of the ring display the protein. Scale bars in all of the AFM images are 100 nm. (3) He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A. E.; Jiang, W.; Mao, C. Nature 2008, 452, 198. (4) Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. Nature 2009, 459, 414. (5) Ke, Y.; Douglas, S. M.; Liu, M.; Sharma, J.; Cheng, A.; Leung, A.; Liu, Y.; Shih, W. M.; Yan, H. J. Am. Chem. Soc. 2009, 131, 15903. (6) Wei, B.; Dai, M.; Yin, P. Nature 2012, 485, 623. (7) Ke, Y.; Ong, L. L.; Shih, W. M.; Yin, P. Science 2012, 338, 1177. (8) Martin, T. G.; Dietz, H. Nat. Commun. 2012, 3, 1103. (9) Yan, H.; Park, S. H.; Finkelstein, G.; Reif, J. H.; LaBean, T. H. Science 2003, 301, 1882. (10) Yin, P.; Hariadi, R. F.; Sahu, S.; Choi, H. M.; Park, S. H.; Labean, T. H.; Reif, J. H. Science 2008, 321, 824. (11) Wilner, O. I.; Orbach, R.; Henning, A.; Teller, C.; Yehezkeli, O.; Mertig, M.; Harries, D.; Willner, I. Nat. Commun. 2011, 2, 540. (12) Dietz, H.; Douglas, S. M.; Shih, W. M. Science 2009, 325, 725. (13) Han, D.; Pal, S.; Nangreave, J.; Deng, Z.; Liu, Y.; Yan, H. Science 2011, 332, 342. (14) Zhang, J.; Liu, Y.; Ke, Y.; Yan, H. Nano Lett. 2006, 6, 248. (15) Sharma, J.; Chhabra, R.; Cheng, A.; Brownell, J.; Liu, Y.; Yan, H. Science 2009, 323, 112. (16) Schreiber, R.; Kempter, S.; Holler, S.; Schuller, V.; Schiffels, D.; Simmel, S. S.; Nickels, P. C.; Liedl, T. Small 2011, 7, 1795. (17) Armani, D. K.; Kippenberg, T. J.; Spillane, S. M.; Vahala, K. J. Nature 2003, 421, 925.
scaffolds that display many target probes or recognition molecules through very few modifications, which is essential for applications such as targeted drug delivery and for sensitive biomolecule-detection. In summary, we describe the assembly of discrete DNA ring structures composed of repeating tubular units using only 8 or 12 unique DNA strands that are 20−30 nts long. For all the designs examined here, rings of nearly uniform size were achieved with mean diameters that are comparable to theoretically predicted ones. These rings are made from small repeating units that can be used to display many copies of target molecules on selected helices and may be used as scaffold for nanomaterial16 such as optical cavities17 and nanomedical applications.
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ASSOCIATED CONTENT
S Supporting Information *
Methods and materials, DNA sequences, additional AFM images, gels, and statistical analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: (H.Y.)
[email protected]; (Y.L.)
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by a National Science Foundation Grant 1104373, an Office of Naval Research grant N000140911118, an Army Research Office Grant W911NF11-1-0137 to H.Y. and Y.L., and an Army Research Office MURI award W911NF-12-1-0420 to H.Y. H.Y. is supported by the Presidential Strategic Initiative Fund from Arizona State University.
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REFERENCES
(1) Winfree, E.; Liu, F.; Wenzler, L. A.; Seeman, N. C. Nature 1998, 394, 539. (2) Rothemund, P. W. Nature 2006, 440, 297. 1866
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