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Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication

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Science  09 Dec 2005:
Vol. 310, Issue 5754, pp. 1661-1665
DOI: 10.1126/science.1120367

Abstract

Practical components for three-dimensional molecular nanofabrication must be simple to produce, stereopure, rigid, and adaptable. We report a family of DNA tetrahedra, less than 10 nanometers on a side, that can self-assemble in seconds with near-quantitative yield of one diastereomer. They can be connected by programmable DNA linkers. Their triangulated architecture confers structural stability; by compressing a DNA tetrahedron with an atomic force microscope, we have measured the axial compressibility of DNA and observed the buckling of the double helix under high loads.

Three-dimensional (3D) construction by self-assembly requires rigid building blocks such as tetrahedra. DNA is an ideal material for nanofabrication of rigid structures because assembly can be controlled by base-pairing (1) and is relatively inexpensive and simple to execute (2). However, DNA nano-fabrication presents the problem of avoiding unwanted by-products. It is often possible to ensure that the target structure is the one that creates the largest number of Watson-Crick base pairs and is therefore the most stable product. Usually, however, there are many other possible structures that are only slightly less stable. If all component oligonucleotides are simply mixed without precaution, the yield of the target structure can be extremely low, and disordered polymeric structures can form instead. Successful strategies for the synthesis of 2D periodic structures involve a hierarchy of interactions in which preformed building blocks are linked by weaker interactions to form an array (35), but 3D construction is much less well developed.

Polyhedral DNA nanostructures with the connectivities of a cube (6) and of truncated and regular octahedra (7, 8) have been made, each using a different synthetic strategy. Trisoligonucleotidyls—three oligonucleotides connected by a trifunctional linker (9)—have also been reported to form tetrahedra (10). The cube (6) was assembled in solution by ligation of 10 oligonucleotides, in three stages with intermediate purification steps, with 1% yield. The solid-support synthesis of the truncated octahedron (7) allowed greater control of the assembly process: Two halves of an edge could be joined by ligation only after a deprotection step in which a restriction endonuclease was used to cleave two precursor hairpin loops to create overlapping sticky ends. This synthesis, starting with 48 oligonucleotides, took approximately two worker-years; yield was less than 1%. Both the cube and the truncated octahedron were covalently closed catenanes that could not be disassembled without breaking covalent bonds: In designing the octahedron (8), this robust design principle was sacrificed to permit assembly by folding. The principal component of the octahedron, a 1.7-kb oligonucleotide synthesized using 64 synthetic oligonucleotides and amplified by cloning, was designed to have branched secondary structure; the octahedron was formed when branches folded and were bound together by intramolecular paranemic interactions (11).

The junctions that form the vertices of these 3D nanostructures are flexible. DNA nanostructures with triangulated architectures may be capable of resisting deformation, but their mechanical properties have not been measured. Rigidity is not enough to ensure that a DNA polyhedron has a robust and well-defined structure; it is also necessary to select one of the two possible diastereomers (enantiomers with respect to the identities of their vertices) that satisfy the pattern of connectivity imposed by the design of hybridization interactions. Discrimination between diastereomers of DNA polyhedra has yet to be demonstrated, and the stereoselectivity of the syntheses described above is unknown.

We have synthesized a family of DNA tetrahedra that have been designed to self-assemble in a single step in only a few seconds. A single diastereomer can be synthesized with yields as high as 95%. We demonstrate their versatility as building blocks for 3D nanofabrication by assembling one regular and nine different irregular tetrahedra and by connecting them with programmable DNA linkers. We then use atomic force microscopy (AFM) to image the tertiary structure of individual tetrahedra and to demonstrate their rigidity, which we exploit to measure the response of DNA to axial compression.

The DNA tetrahedron is designed to be mechanically robust; it consists of rigid triangles of DNA helices covalently joined at the vertices (Fig. 1A) (12). The four component oligonucleotides each run around one face and hybridize to form the double-helical edges. Four edges contain nicks (i.e., breaks in the DNA backbone) where the 5′ and 3′ ends of an oligonucleotide meet. At each vertex, adjacent edges are attached through single, unpaired “hinge” bases. In contrast to the challenging syntheses of DNA cubes (6) and octahedra (7, 8), the synthesis of tetrahedra is extremely simple: All four oligonucleotides are combined in equimolar quantities in hybridization buffer at 95°C and then cooled to 4°C in 30 s (13).

Fig. 1.

DNA tetrahedra. (A) Design of a DNA tetrahedron formed by annealing four oligonucleotides. Complementary subsequences that hybridize to form each edge are identified by color. (B) Two views of a space-filling representation of a 3×20/3×30-bp tetrahedron. The backbone of each oligonucleotide is indicated by a single color. (C) AFM image showing several tetrahedra on a mica surface. (D) AFM images, recorded with ultrasharp tips, of four tetrahedra; the three upper edges are resolved.

Tetrahedra form with ∼95% yield and migrate as single bands on a nondenaturing electrophoresis gel (fig. S1) (13). A covalently closed catenane may be produced by enzymatic ligation of the four nicks in the DNA backbone. We believe that the designed hierarchy of interactions between oligonucleotides contributes to the high efficiency of this one-step synthesis. We expect hybridization between oligonucleotides 1 and 2, and also 3 and 4, to form the stable, unnicked edges B and E (Fig. 1A) to occur first as the solution temperature falls. Other edges can then form cooperatively; once the formation of any other edge has linked these pairs to form a four-strand complex, all further hybridization interactions required to complete the tetrahedron are intramolecular and are therefore expected to be faster than competing intermolecular interactions that would form larger complexes. The positions of the nicks are such that none of these intramolecular interactions is substantially hindered by bonds already formed.

The tetrahedra imaged by AFM in Fig. 1, C and D, were designed to have three 30–base pair (bp) edges meeting at one vertex and three 20-bp edges bounding the opposite face (a molecular model is shown in Fig. 1B). They are expected to bind to a surface in one of two orientations, with heights of ∼10.5 nm if resting on the small face and ∼7.5 nm if resting on any of the other three faces. Figure 1C, recorded with a tip 20 nm in radius, shows several objects with heights consistent with the two orientations. Figure 1D shows high-resolution images, obtained using ultrasharp tips with radii of only 2 to 3 nm, that resolve the three upper edges of individual tetrahedra.

To confirm that our constructs had the topology of a tetrahedron, we used selective enzymatic ligation and digestion. Incubation with T4 DNA ligase leads to ligation (covalent closure) of the nicks where the 5′ and 3′ ends of an oligonucleotide are held together in the middle of an edge, but only if the 5′ end is prepared with a terminal phosphate group. Sixteen regular 20-bp tetrahedra were formed with every combination of ligated and unligated nicks. In the denaturing gels shown in Fig. 2, A to C, linear oligonucleotides dissociated and only circular, catenated oligonucleotides were constrained to migrate together. According to the design, each ligation should produce a circular oligonucleotide and all circles produced by multiple ligations should be catenated with a linking number corresponding to the number of complete helical turns in each edge—in this case, two. The expected bands appeared when one, two, three, and four oligonucleotides were ligated (products of failed ligation were also observed). Digestion with exonuclease III, which can hydrolyze duplex DNA from a free 3′ end, confirmed that they contained circular oligonucleotides (Fig. 2C, lane 3). The topology of the corresponding single-, double-, triple-, and quadruple-linked circles can be described using Conway's notation (14) as (∞), (–4), (4,4,4), and (6**13.4.13.4.13.4), respectively (fig. S2) (13). These results are consistent with the topology of the designed structure.

Fig. 2.

Topological and structural analysis of a 20-bp regular tetrahedron. (A and B) Denaturing gels showing products of all possible combinations of ligated and unligated nicks. Control lanes contain oligonucleotides of the same length as the four components of the tetrahedron: linear (lanes A1 and B1), circular (lanes A2 and B2), and double (lanes A3 and B3) and triple (lane B4) linked circles. (C) Fully ligated tetrahedron (lane 1) after gel purification (lane 2) and Exo III digestion (lane 3). (D and E) Edge digestions of a fully ligated tetrahedron on a native gel. (D) Lanes 1 to 6, single cuts; lane 7, uncut tetrahedron. (E) Lanes 2 to 16, double cuts; lanes 1 and 17, uncut tetrahedron. Lane M, 50-bp ladder. See (13) for synthesis of markers and for keys to ligated oligonucleotides and edge-cutting enzymes.

Because each edge of the tetrahedron has a different base sequence, sequence-specific enzymatic digestion can be used to provide further confirmation of the tetrahedron's tertiary structure. Each edge was designed to contain a different restriction sequence that may be digested (cut) by one of six restriction endonucleases. The effects of edge digestion on a fully ligated tetrahedron are shown using native gels in Fig. 2, D and E. None of the six possible single-edge cuts, not even the blunt cut produced by Alu I (lane 1), had a measurable effect on the tetrahedron's mobility (Fig. 2D). We conclude that the tetrahedron's tertiary structure is particularly stable. The products of each of the 15 possible double-edge digests are shown in Fig. 2E. Three of the cuts created a band with lower mobility than the uncut band; the remaining 12 cut bands had higher mobility. The two groups correspond to two distinct ways of cutting the tetrahedron twice: Higher mobility bands were created by cuts on adjacent edges and lower mobility bands by cuts on opposite edges, confirming the designed relations between edges.

Our assembly method is extremely flexible. Figure 3A shows two series of tetrahedra made with four 20-bp edges; a fifth edge of 20 or 10 bp, respectively; and a sixth edge, opposite the fifth, that varied in length between 10 and 30 bp. Each synthesis resulted in a single-band product whose mobility decreased with increasing edge length. We can also adapt the design to introduce nicks into all six edges (fig. S3) (13); single-stranded overhangs at these nicks could be used to create sticky ends to join tetrahedra to make 3D structures.

Fig. 3.

Versatility and stereoselectivity of tetrahedron synthesis. (A) Tetrahedra with five 20-bp edges and one edge of 10 bp (lane 1), 15 bp (lane 2), 20 bp (lane 3), 25 bp (lane 4), or 30 bp (lane 5). Tetrahedra with four 20-bp edges, one 10-bp edge, and an opposite edge of 10 bp (lane 6), 15 bp (lane 7), 20 bp (lane 8), 25 bp (lane 9), or 30 bp (lane 10). For both series the tetrahedra in the first and last lanes are illustrated by 3D models; the edge that is varied is marked with an arrow. (B) Linking experiments demonstrating stereoselectivity. A linking strand may join two 5×20/1×30-bp tetrahedra by hybridizing in 10-bp single-stranded gaps in both long edges. There are two possible diastereomers of a DNA tetrahedron. Four gap positions, two in each strand forming the edge, were designed such that the linker would emerge on the outside of one diastereomer, accessible for further hybridization (left panel), and on the inside of the other, hindering further hybridization (right panel). A strong dimer band is observed in only the two cases consistent with the presence of the diastereomer, in which the major groove of each helix faces inward at the vertices. See (13) for detailed information on structures. Lane M, 50-bp ladder.

We have investigated an alternative linking strategy based on the incorporation of a single-stranded gap in a tetrahedron edge (Fig. 3B); oligonucleotides containing two subsequences, each capable of hybridizing in a gap, can be used to link tetrahedra in a programmable manner. A linking strand containing two identical subsequences joins preformed tetrahedra to create homodimers as expected (Fig. 3B, lanes 2 and 4) (fig. S4) (13). We have also used linkers incorporating two different binding sequences to create heterodimers (fig. S5) (13).

Dimer formation was used to investigate the stereoselectivity of the synthesis. The gapped edge is not free to rotate: The position of the gap along the edge determines the azimuthal position of the free end of the hybridized linker, and thus whether it can reach and hybridize with another tetrahedron. Two gap positions were designed, one for each of the two strands forming the edge, such that the linking strand would project away from the center of one diastereomer but into the center of the other (Fig. 3B, outer panels). In the latter configuration, the linker is expected to be inaccessible. Controls with gaps translated by five bases (half a helical turn) were designed to have the opposite linker orientation. The results of linking experiments for a tetrahedron with 5×20/1×30-bp edges (Fig. 3B) are consistent with the presence of a large excess of the diastereomer in which the major groove of each helix faces inward at each vertex, indicating a significant difference between the formation rates or stabilities of the two possible diastereomers. Stereoselective synthesis, in combination with structural rigidity, ensures that the relative coordinates of any part of the structure can be defined with near-atomic accuracy, an essential property of a nanostructure to be used as a building block for molecular nanofabrication.

We used these structurally braced tetrahedra to investigate the behavior of DNA under compression. Although DNA under tension has been widely studied (1518), DNA strands of micrometer length buckle at extremely low forces. Measurement of the response of DNA to large compressive loads could help to resolve the current controversy over the nature of structural changes associated with rare large-angle deformations of the double helix (19, 20). To measure the mechanical response of a single tetrahedron directly, we used an AFM tip as a sensitive force transducer. The tip was centered over a tetrahedron located in imaging mode and was then moved toward the surface while recording force. Compression curves for seven distinct 3×20/3×30-bp tetrahedra, as imaged in Fig. 1, are shown in Fig. 4. For forces up to ∼100 pN, the response was approximately linear and reversible (Fig. 4, inset) with an average force constant of 0.18 (±0.07) N m–1. At higher forces, the response was nonlinear and varied from tetrahedron to tetrahedron; tetrahedra generally softened suddenly and deformed irreversibly at a load between 70 and 200 pN.

Fig. 4.

Compression of single DNA tetrahedra. Compression curves show linear elastic response up to a load of 0.1 nN. At higher forces, most tetrahedra deform irreversibly. Offsets were adjusted to overlap the linear parts of the seven curves. Inset: Reversibility of the elastic response of a typical tetrahedron.

To model the compressibility of a DNA tetrahedron, we treat its edges as elastic rods pinned (freely hinged) at the vertices. The calculated response of a 3×20/3×30-bp tetrahedron to a compressive load applied between the top vertex and the surface supporting the bottom face is approximately the same for both orientations and is dominated by axial compression of the upstanding edges. The calculated force-displacement (F-d) curve is approximately linear up to a critical load at which the tetrahedron buckles. The boundary conditions at the bottom face have a small effect on the response: If the bottom vertices are not fixed but allowed to slide on the surface, then the bottom edges stretch and the overall stiffness of the construct is reduced by ∼3% and ∼13% for the tall and short orientations of the tetrahedron, respectively. From the gradient of the linear part of the measured F-d curve, we infer an elastic modulus of Kc = 0.7 (±0.3) nN for one DNA double helix in compression. In the linear response regime, elastic moduli measured by extension and compression should be equal; our value for Kc is near that of the elastic modulus of DNA in tension, Ke ∼1.1 nN, obtained by fitting the force-extension curves of DNA duplexes (17, 18). Our direct measurement of the axial elastic response of DNA in compression was made possible by the braced structure of the tetrahedron that enabled a short DNA helix to bear a compressive load without bending or tilting.

We can use our measured elastic modulus to estimate the load at which we would expect the edges of a tetrahedron to buckle. If a DNA duplex is modeled as a uniform cylinder of radius r = 1 nm (21), then the critical compressive force in an edge at which we would expect Euler instability is Fc = π2r2K/(2υl)2, where K is the elastic modulus, l is the edge length, and υ is a numerical factor that depends on the boundary conditions at the vertices. If the vertices are pinned, then υ = 1; if the orientations of the edges at the vertices are fixed, then υ = ½. The corresponding AFM tip loads lie in the range from 50 to 300 pN, which is consistent with the range of loads at which tetrahedra were observed to soften suddenly. Our observations of the failure of tetrahedra under high load can thus be explained on the basis of the traditional model of uniform DNA bending (20); this result is consistent with our interpretation that the linear part of the F-d curve is caused by pure axial compression of the tetrahedron's upstanding edges before buckling occurs.

The structural changes associated with DNA bending are the subject of controversy. Suggestions that sharp bends due to local melting or kinking (22) are observed in DNA cyclization experiments (19, 23, 24) are countered by measurements that indicate that the probability of such kinks is very low (20). Extended observation of tetrahedra under compression may be a useful method for investigating the energy and sequence dependence of inhomogeneous bending and of the effects of compressive strain on DNA-protein interactions.

Supporting Online Material

www.sciencemag.org/cgi/content/full/310/5754/1661/DC1

Materials and Methods

SOM Text

Figs. S1 to S5

References

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