DNA Nanocages: Self-Assembly from Star Motifs for 3D Nanostructures, Notas de estudo de Engenharia de Produção

Baixe DNA Nanocages: Self-Assembly from Star Motifs for 3D Nanostructures e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! PAPER 143/14 www.rsc.org/faraday_d | Faraday Discussions 1 5 10 15 20 25 30 35 40 45 50 55DNA self-assembly: from 2D to 3D Chuan Zhang,a Yu He,a Min Su,b Seung Hyeon Ko,a Tao Ye,a Yujun Leng,b Xuping Sun,a Alexander E. Ribbe,a Wen Jiangb and Chengde Mao*aReceived 16th March 2009, Accepted 6th April 2009 First published as an Advance Article on the web ????? DOI: 10.1039/b905313cThis paper describes our recent efforts on the self-assembly of three-dimensional (3D) DNA nanostructures from DNA star motifs (tiles). DNA star motifs are a family of DNA nanostructures with 3, 4, 5, or 6 branches; they are named as 3-, 4-, 5-, 6-point-star motifs, respectively. Such motifs are programmed to further assemble into nanocages (regular polyhedra or irregular nanocapsules) with diameters ranging from 20 nm to 2 mm. Among them, DNA nanocages derived from 3-point-star motif consists of a group of regular polyhedra: tetrahedra, hexahedra (or cubes), dodecahedra and buckyballs (containing 4, 8, 20, and 60 units of the 3-point-star motif, respectively). An icosahedron consists of twelve 5-point-star motifs and is similar to the shapes of spherical viruses. 6-point-star motifs can not assemble into regular polyhedra; instead, some sphere-like or irregular cages with diameters about 1–2 mm will form. Similar large cages can also assemble from the 5-point-star motif when the DNA concentrations are higher than those for assembling regular icosahedra. In our study, we have identified several important factors for assembly of well-defined 3D nanostructures, including the concentration, the flexibility, and the arm length of the DNA tiles and the association strength between the DNA tiles.1. Introduction Beyond the genetic interest, DNA has been shown as a superb molecular system in self-assembly towards bottom-up nanofabrication.1–3 It has many attractive proper- ties: the excellent capability of molecular recognition, the well-defined secondary structure (duplex), reasonable chemical stability, and readily commercial avail- ability. These properties together endow DNA molecules with a great potential to serve as ‘‘building blocks’’ for the preparation of nanostructures. In structural DNA nanotechnology, DNA motifs (tiles) are designed and investi- gated as building blocks to fabricate nanostructures. For example, a range of DNA motifs including double crossover (DX) and multiple-crossover motifs, star motifs, triangle and DX triangle motifs, and parallelogram motifs, have been introduced to construct various DNA two-dimensional (2D) arrays and non-periodic 2D patterns.4–18 However, the investigation of DNA 3D nanostructures is quite limited.19–27 In the early 90s, DNA 3D nanostructures with the connectivities of a cube19 and of a truncated octahedron20 were constructed by Seeman and his coworkers. Limited by characterization methods (denaturing gel electrophoresis), such 3D structures had to be covalently linked, which required the authors to useaDepartment of Chemistry, Purdue University, West Lafayette, Indiana, 47907, USA. E-mail: mao@purdue.edu bMarkey Center for Structural Biology and Department of Biological Sciences, Purdue University, West Lafayette, Indiana, 47907, USA ART  B905313C This journal is ª The Royal Society of Chemistry 2009 Faraday Discuss., 2009, 143, 1–13 | 1 1 5 10 15 20 25 30 35 40 45 50 55stepwise synthesis via enzymatic ligation. Successive steps of ligation and purifica- tion, unfortunately, greatly increased the workload and resulted in a very low overall synthetic yield. With the introduction of new characterization tools [cryogenic elec- tron microscopy (cryoEM) and atomic force microscopy (AFM)], one was able to visualize non-covalently associated DNA structures by direct imaging. Starting from 2004,21 strategies of one pot self-assembly were used to assemble DNA 3D nanostructures. For example, Turberfield and his coworkers used four oligonucleo- tides to assemble DNA tetrahedra;22 the resulting structures were characterized by AFM. In another elegant example, Shih and his coworkers folded a long single DNA strand into pre-designed, branched, secondary structures, which further folded into an octahedon through intra-complex paranemic interactions with the help of a number of short DNA helper strands.21 Essentially, this is related to the so-called ‘‘DNA origami’’ approach.16 The resulting octahedral structures were clearly shown by cryoEM characterization. This work together demonstrated that DNA polyhedra could be readily assembled from synthetic DNA molecules. Though tetrahedra, cubes, octahedral, and truncated octahedra are highly symmetric, those reported DNA 3D objects do not really possess these symmetries if viewed at the level of DNA sequences. Consequently, every object requires many unique DNA strands. As the objects become larger and more complicated, the number of DNA strands with unique sequences quickly increases. It is not easy to apply the above mentioned methods to construct complicated polyhedra, which require too many unique DNA strands. Thus, while these studies are elegant and exciting, they do not provide a general and simple route to fabricate DNA 3D nano- structures. In biology, many complex nanostructures with various biological func- tions assemble in different ways. For instance, the outer protein shells (capsids) of spherical viruses have highly symmetric, icosahedral structures. They are composed of many copies of identical protein subunits, which associate with each other via non-covalent bonding. The complexity and robustness of viral capsids indicateFig. 1 DNA nanocages self-assembled from DNA star motifs. ART  B905313C 2 | Faraday Discuss., 2009, 143, 1–13 This journal is ª The Royal Society of Chemistry 2009 Fig. 3 Visualization of the self-assembled DNA polyhedra by cryogenic transmission electron microscopy (cryoTEM). (top panel) tetrahedra, (middle panel) dodecahedra, and (bottom panel) buckyballs. (a) A representative image. White boxes indicate the DNA particles. (b) Comparison between raw images of individual particles (left) and the corresponding ART  B905313C 1 5 10 15 20 25 30 35 40 45 50 55 This journal is ª The Royal Society of Chemistry 2009 Faraday Discuss., 2009, 143, 1–13 | 5 1 5 10 15 20 25 30 35 40 45 50 55CryoEM (Fig. 3, middle panel) showed dodecahedral-shaped objects of the ex- pected size (in the boxed area). All particles with the right size were picked up for single particle 3D reconstruction. The icosahedral symmetry was imposed during the reconstruction, resulting in a well-defined dodecahedral structure (at a resolution of 2.5–3.0 nm). The individual, raw cryoEM particles matched with the computer- generated model projections very well.Buckyballs 3-point-star tiles can also assemble into Buckminsterfullerenes (or buckyballs), a type of highly symmetric polyhedron. A buckyball contains 60 vertexes, 90 edges, and 32 faces (12 pentagons and 20 hexagons). Assembling DNA buckyballs and dodecahedra require exactly the same DNA strands. To selectively assemble these structures, the DNA concentration has to be carefully controlled. At a high DNA concentration (500 nM), the 3-point-star DNA tiles (the central, single-stranded loops are 3 bases long) readily assemble into the large buckyball structures rather than small dodecahedra. The DLS measurement indicates that the DNA assemblies have an apparent hydrodynamic radius of 42.2  4.0 nm, close to the calculated radius of the buckyball model (41 nm). However, the polydispersity of the assembled DNA buckyballs is significantly higher than those of DNA tetrahedra and dodeca- hedra. Under AFM imaging, the DNA assemblies appear as uniform-sized, round, collapsed DNA nanostructures with a diameter of 110 nm. Round-like DNA structures can be seen in the raw CryoEM images (Fig. 3, bottom panel) and a bucky- ball structure has been obtained from reconstruction based on hand-picked parti- cles. Compared to tetrahedra and dodecahedra, the size distribution of the particles in the cryoEM images is much worse than that from the AFM images. This phenomenon is likely due to larger assemblies being easier to deform or break up, which lowers the quality of the reconstructed map. The first three 3D objects have a common feature in their design. Each pseudo- continuous DNA duplex of the edge in the final polyhedra is 4 turns (42 bases) long after sticky end hybridization. In such a design, all component DNA tiles are facing the same direction and any intrinsic curvatures of the tiles would add up toward the same direction to promote the formation of closed structures. Mean- while, the DNA concentration plays a key role during the self-assembly. Self- assembly relies on inter-tile interactions. High DNA concentrations favor large assemblies in the same design such as buckyballs, and low DNA concentrations will promote small assemblies such as dodecahedra. The desired products dominate in self-assembly only in a certain range of DNA concentration.Cubes (or hexahedra) When carefully balancing the flexibilities and the rigidities of the DNA motifs and controlling the DNA concentrations, 3-point-star motifs can selectively assemble into tetrahedra, dodecahedra, or buckyballs. However, the cube structure, a simple polyhedron that contains eight 3-point-star tiles, has never been found in the study discussed above. It implies that cube structures cannot be obtained by simply changing the concentration and the flexibility of the DNA tiles. To overcome this problem, we introduced another strategy to the self-assembly of 3D DNA nanostructures.30 This strategy exploits the helical nature of the DNA double helix structure. When being separated by an odd number of half helical turns, two objects along a DNA duplex will be on the opposite sides of the DNA duplex and are related by a two-fold rotational symmetry. To assemble cubes, each edgecomputer-generated model projections (right). (c) Three views of the DNA polyhedral structure reconstructed from cryoTEM images. (Replicated from ref. 28). ART  B905313C 6 | Faraday Discuss., 2009, 143, 1–13 This journal is ª The Royal Society of Chemistry 2009 Fig. 4 Self-assembly of DNA cubes from 3-point-star tiles. (left) Assembly scheme. Note that the separation of any two adjacent tiles is 4.5 DNA helical turns. Any two interacting tiles (shadowed with different colors) are related by a two-fold rotational axis, indicated by arrowed black lines. (right) cryoEM characterization of the DNA cubes. (a) a raw cryo-EM image of the DNA sample; (b) close-up view of several representative raw particles from (a) (upper row) and their corresponding computer-generated model projections (lower row); (c) the 3D reconstruc- tion maps of the DNA cube, reconstructed by imposing a tetrahedron symmetry. (Replicated from ref. 30). 1 5 10 15 20 25 30 35 40 45 50 55is designed to be 4.5 turns long (Fig. 4, left). The odd number of DNA helical turns leads to any two adjacent tiles to face to different sides of the DNA tile plane. Thus, any closed rings must contain an even number of tiles: half of the tiles face inward and the other half face outward. The smallest polyhedron that meets this require- ment is a cube; which suggests that the 3-point-star motif at sufficiently low concen- trations will assemble into cubes. The DNA cubes were assembled at a DNA concentration of 50 nM. Most of the DNA tiles were incorporated into a large, well-defined, molecular complex, which appeared as a sharp band in polyacrylamide gel electrophoresis. The assembly yield was 82% as estimated from the gel. DLS experiment indicated that the DNA complex had an apparent hydrodynamic radius of 16.0  2.8 nm. This value agreed well with the radius of the circumscribed sphere of the DNA cube (radius: 16.5 nm). Under cryoEM imaging (Fig. 4, right), most observed particles were consistent with the 2D projections of cubes at the expected size (the cube edge is 15 nm long). With experimentally observed particles, a DNA cube structure at 2.9 nm resolution was revealed by single particle 3D reconstruction in which tetrahedron symmetry was imposed. By comparing the 2D projections of the reconstructed cube model and the raw images with similar orientations, clear similarities confirmed that the self-assembled DNA complex indeed had a cube structure.3. DNA nanocages assembled from 5-point-star motifs The planar tiling theory predicts that objects with 5-fold rotational symmetry can not assemble into regular (2D) lattices to completely tile a plane. So in previous studies of 2D self-assembly, 5-point-star motifs are avoided. However, the obstacle for 2D crystal growth might be a benefit for 3D self-assembly. The 5-point-star motifs have been shown to be excellent building blocks for DNA 3D nanocages inART  B905313C This journal is ª The Royal Society of Chemistry 2009 Faraday Discuss., 2009, 143, 1–13 | 7 Fig. 7 AFM characterization of DNA nanocages assembled from 5-point-star tiles with different strengths of sticky-end association. (a) 1-GC-basepair sticky ends; (b) 2- GC-basepair sticky ends; (c) 3-GC-basepair sticky ends; (d) 4-GC-base pair sticky ends. AFM images are taken in air. Each pair of AFM images shows the DNA structures at two imaging scales. 1 5 10 15 20 25 30 35 40 45 50 55cages land onto AFM substrate (mica surfaces), they collapse into double-layer structures due to strong interactions between DNA and mica surfaces. After several successive AFM scans, the top layers are occasionally scratched out by the AFM probes and only the monolayers are left. For such monolayer samples, the DNA assemblies show regular, periodic arrays, which have a tetragonal symmetry. With 3-GC-basepair sticky-ends, most DNA assemblies are capped nanotube-like structures, indicating an anisotropic growth during the self-assembly process. The origin of the anisotropy is unclear. One speculation is that different bending orientations of the five arms break the growth symmetry. The complexes with 2-GC-basepair sticky-ends show similar morphologies to the complexes with 3-GC-basepair sticky-ends, but with a shorter tube length. With 1-GC-basepair sticky-ends, the DNA complexes are not as stable as others because of the weak association. After washing, cracks can be found in the nanocages under AFM imaging. The strength of sticky-end association clearly influences the stabilities of the DNA nanocages as well. The DNA nanocages are stable at low temperatures and disassociate at high temperatures. This process can be monitored by DLS measurement (Fig. 8). The hydrodynamic radii of the DNA nanocages areART  B905313C 10 | Faraday Discuss., 2009, 143, 1–13 This journal is ª The Royal Society of Chemistry 2009 Fig. 8 DLS monitoring of the dissociation of DNA nanocages. (top) The DLS data at different temperatures for DNA cages assembled from 5-point-star tiles with 1-GC sticky- ends. (bottom) The melting temperatures of DNA nanocages assembled from different 5-point-star tiles determined by DLS. DNA concentration: 1 mM. 1 5 10 15 20 25 30 35 40 45 50 55much larger than those of the individual tiles. As shown in Fig. 8, higher GC contents in the sticky-ends results in more stable DNA nanocages and higher melting temperatures.4. DNA nanocages assembled from 6-point-star motifs A 6-point-star motif is the most branched star motif investigated so far. It can asso- ciate into nanocages with sphere-like or irregular shapes. Their sizes range from several hundred nanometers to 2 mm as determined by AFM imaging (Fig. 9). The sticky-end effect exists for the self-assembly of 6-point-star tiles as that of 5-point-star tiles. Under the same assembly conditions, the 3-GC tiles assemble into stable nanocages that collapse into double-layer discs (less than 2 mm wide) upon deposition onto mica surfaces. For 2-GC tiles, less cage structures are found. Instead, large 2D monolayers (>10 mm) can be found under AFM imaging. When further reducing the GC content to 1-GC tiles, mica surfaces are fully covered by small monolayer 2D arrays with domain sizes typically less then 2 mm.5. Conclusions In this paper, we have discussed all the DNA nanocages derived from DNA star tiles in our recent research. The DNA cages have sizes ranging from 20 nm–2 mm. In such 3D self-assembling processes, balancing the rigidity and the flexibility of the DNA tiles is the key idea to make nanostructures in the desired size. The DNAART  B905313C This journal is ª The Royal Society of Chemistry 2009 Faraday Discuss., 2009, 143, 1–13 | 11 Fig. 9 Self-assembly of DNA nanocages from 6-point-star motifs. (top) Scheme of the DNA self-assembly. (bottom) AFM images of the DNA nanocages with different association strengths. 1 5 10 15 20 25 30 35 40 45 50 55concentration is another important factor that influences the assembly kinetics and sometimes determines the final morphologies of the assemblies. Future research might include several aspects: (1) To organize nanoparticles or proteins in 3D space using DNA polyhedra as templates; (2) To encapsulate drugs in the DNA cages for delivery or capsulate biomacromo- lecules for single particle studies; (3) To develop a new strategy to fabricate complicated 3D structures and develop responsive DNA cages; (4) To quantitatively study 3D DNA self-assembly in terms of both thermody- namics and kinetics.Acknowledgements This work was supported by the National Science Foundation (CCF-0622093). DLS studies were carried out in the Purdue Laboratory for Chemical Nanotechnology (PLCN). The cryo-EM images were taken in the Purdue Biological ElectronART  B905313C 12 | Faraday Discuss., 2009, 143, 1–13 This journal is ª The Royal Society of Chemistry 2009