Designer DNA Nanoarchitectures

Designer DNA Nanoarchitectures

(Parte 1 de 4)

Current Topics

Designer DNA Nanoarchitectures†

Chenxiang Lin, Yan Liu, and Hao Yan*

Department of Chemistry and Biochemistry and The Biodesign Institute, Arizona State UniVersity, Tempe, Arizona 85287 ReceiVed December 19, 2008; ReVised Manuscript ReceiVed January 16, 2009

ABSTRACT: Naturally existing biological systems, from the simplest unicellular diatom to the most sophisticated organ such as the human brain, are functional self-assembled architectures. Scientists have long been dreaming about building artificial nanostructures that can mimic such elegance in nature. Structural DNA nanotechnology, which uses DNA as a blueprint and building material to organize matter with nanometer precision, represents an appealing solution to this challenge. On the basis of the knowledge of helical DNA structure and Watson-Crick base pairing rules, scientists have constructed a number of DNA nanoarchitectures with a large variety of geometries, topologies, and periodicities with considerably high yields. Modified by functional groups, those DNA nanostructures can serve as scaffolds to control the positioning of other molecular species, which opens opportunities to study intermolecular synergies, such as protein-protein interactions, as well as to build artificial multicomponent nanomachines. In this review, we summarize the principle of DNA self-assembly, describe the exciting progress of structural DNA nanotechnology in recent years, and discuss the current frontier.

The central task of nanotechnology is to control motions and organize matter with nanometer precision. To achieve this, scientists have intensively investigated a large variety of materials, including inorganic materials (e.g., carbon nanotubes) (1), organic molecules (2) and biological polymers (e.g., peptides, RNA, and DNA) (3, 4), and different methods that can be sorted into so-called “bottom-up” and “top-down” approaches. Among all of the remarkable achievements made, the success of DNA self-assembly in building programmable nanopatterns attracts broad attention and holds great promise for building novel designer nanoar- chitectures (4). DNA is an excellent nanoconstruction material because of its inherent merits. First, the rigorous Watson-Crick base pairingmakes the hybridizationbetween DNA strands highly predictable. Second, the structure of the B-form DNA double helix is well-understood; its diameter and helical repeat have been determined to be ∼2 and ∼3.4 nm (i.e., ∼10.5 bases), respectively, which facilitates the modelingof even the most complicatedDNA nanostructures. Third, DNA possesses combined structural stiffness and flexibility. The rigid DNA double helices can be linked by relatively flexible single-stranded DNA (ssDNA)1 to build stable motifs with desired geometry. Fourth, modern organic chemistry and molecular biology have created a rich toolbox for readily synthesizing, modifying, and replicating DNA† This work was supported by grants from the National Science Foundation(NSF), the Army Research Office (ARO), and the Technol- ogy and Research InitiativeFund from Arizona State Universityto Y.L. and by grants from NSF, ARO, the Air Force Office of Scientific Research, the Office of Naval Research, and the National Institutes of Health to H.Y.

* To whom correspondence should be addressed. E-mail: Phone: (480) 727-8570. Fax: (480) 965-2747.

1 Abbreviations: ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; 1D, one-dimensional; 2D, two-dimensional; 3D, threedimensional;DX, doublecrossover;TX, triplecrossover;PX, paranemic crossovers; PCR, polymerase chain reaction; AuNP, gold nanoparticle; RCA, rolling circle amplification.

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molecules.Finally,DNA is a biocompatiblematerial,making it suitable for the constructionof multicomponentnanostructures made from heterobiomaterials.

The simplestexample of DNA self-assemblycan be found in almost all living forms in nature: two complementary ssDNAmoleculesspontaneouslyhybridizetogetherand form a double-stranded DNA (dsDNA) molecule. This process is driven by a number of noncovalent interactions such as hydrogen bonding, base stacking, electrostatic forces, and hydrophobicinteractionsand strictlyobeysthe Watson-Crick base pairing rules. Artificially designed DNA self-assembly is utilized by molecular biologists in gene cloning. In this case, the genomic DNA of interest is extended at both ends to have ssDNA overhangs (sticky ends) complementary to the sticky ends generated by restriction enzymes on the vector; the genomic DNA can then merge into the digested vector, and the nick can be sealed by DNA ligase. However, if more complicated nanostructures are desired, one must employ branched DNA motifs to extend the DNA selfassembly into the second, or even third, dimension. Such motifs have also been found in nature: the DNA replication fork represents a three-way junction motif, and the Holliday (four-arm) junction motif exists as the central intermediate of DNA recombination. In 1982, N. Seeman first envisioned the possibility of combining branched DNA molecules bearing complementary sticky ends to construct twodimensional (2D) arrays (5). This proposal, which was later experimentally realized, is considered to be the very first stepping stone on the path leading to structural DNA nanotechnology.

Figure1a illustratesSeeman’sinitialproposalof DNA selfassembly. Here, the basic building block (also known as “tile”) for the DNA 2D array is a four-arm-junctioncomplex formed by four ssDNA molecules. In addition to the main body, sticky ends are placed at the termini of each tile with the designated base paring strategy (1 is complementary to 1′ and 2 complementary to 2′). As a result, the tiles can infinitely grow into a periodic 2D array through sticky end association. It is intuitive to imagine expanding such selfassembly to the third dimension using three-, four-, five-, or six-arm junction tiles (Figure 1b, gray tiles). This simple model also illustrates that self-assembly is a hierarchical process: the individual tile formations happen first, and the tile-tile associationsfollow.Consequently,Seemanproposed the potential application of self-assembled DNA nanostructures as scaffolds to regulate the positioning of other macromolecules in three dimensions. As shown in Figure 1b, the protein molecules can be assembled parallel to each other with well-defined spatial spacing, directed by the formation of a periodic 3D DNA lattice. Once realized, these lattices could provide a universal means of crystallizing macromolecules and facilitating subsequent structural determination using X-ray diffraction.

The past decade witnessed the fast evolution of structural

DNA nanotechnology (4, 6-10). A large variety of DNA tiles with different geometries and topologies have been constructed with excellent yield. In general, the creation of a novel DNA motif usually requires the following steps. (1) In structural modeling, physical and/or graphic models are used to help in the designof a new DNA motif.Thesemodels are based on the basic knowledge of DNA helical structures (e.g., helical pitch, diameter, base stacking, phosphodiester bond length, etc.) and are built to give the designer straightforward information (e.g., size, shape, and stability) about the designed architecture. The most important consideration for the structural design is to minimize the free energy of the final DNA complex to promote spontaneous assembly. In other words, all the DNA strands involved should stay “comfortably” in the final structure (i.e., no overstretched bonds, no overbent helixes, etc.). Today, computer programs (e.g., Mfold, GIDEON, Tiamat, and Nanoengineer-1) are designed to aid modeling and free energy predictions (1-14). A complicated structure is deemed “good” or “bad” on the basis of empirical parameters, though even the most experienced designer cannot be assuredof successfulassemblyat this stage.Fortunately,with a large and growing number of DNA nanostructures available, we are now richly equipped to design new structures by adapting, joining, or scissoring known structures. (2) For sequence design, specific sequences are assigned to all ssDNA molecules in the model. A general rule of sequence design is to minimize sequence symmetry in the branched structure to avoid possible undesired pairing between participating strands and mobility of the junction points. It is important to point out that sequence symmetry is only avoided within each individual strand but can be allowed between different strands at the symmetric positions in the same tile. The sequence designing process is now automated by computer programs such as SEQUIN (15), Tiamat (13), and Uniquimer (16). It is worth mentioning that these programsare designedto break sequencesymmetrythroughout the whole DNA construct. When symmetry is desired within the system, the sequence for each DNA strand should be designed separately. (3) For experimental synthesis of the DNA nanostructure, the oligonucleotides with designated sequences are synthesized by a DNA synthesizer, purified

FIGURE 1: Principle and application of DNA self-assembly, as proposed by N. Seeman. (a) Principle of DNA self-assembly: combining branched DNA nanostructures with sticky ends to form 2D arrays. Arabic numbers indicate base pairing strategies between sticky ends (1 is complementary to 1′, etc.). (b) Protein crystallization templated by DNA 3D self-assembly.

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via electrophoresisor chromatography,mixed together at the stoichiometricmolar ratio in a near-neutral buffer containing divalent cations (usually Mg2+), heated to denature, and then gradually cooled to allow the ssDNA molecules to find their correct partners and adopt the most energy favorable conformation (i.e., self-assembly). (4) For characterization of the DNA nanostructure, a number of assays can be used to test whether the desired nanostructure forms as designed. The most commonly used methods include nondenaturing gel electrophoresis (tests the integrity of individual tiles), Ferguson study (suggeststhe shape and size of the tiles) (17), hydroxyl radical autofootprinting (determines the junction point)(18), and atomicforce microscopy(AFM) and electron microscopy (EM) (both directly visualize the formation of DNA lattices or large 3D constructions).

RepresentativeDNA tiles and correspondingperiodic selfassembly results are summarized in Figure 2. Closely related to Seeman’s initial proposal, Mao et al. constructed parallelogram DNA junctions (Figure 2a) by covalently joining four Holliday junctions together (19). These tiles can self- assemble into 2D arrays with rhombus-shaped cavities. Applying the concept of “tensegrity” and taking advantage of the natural conformation of the Holliday junction, Mao’s group constructed a triangle tile composed of three Holliday junctions (20). Seeman and colleagues first created a series of DNA double-crossover (DX) motifs by joining two parallel helices together through strand exchange (21). These DX moleculeswere later modifiedto carry properstickyends and successfullyself-assembledinto DNA 2D arrays without observable cavities (2). Using a similar principle, multicrossover molecules, including triple crossover (23) and four-, eight-, and 12-helix planar tiles (24, 25), were synthesized to self-assemble into either DNA nanotubes or 2D lattices. Yan and Labean reported the construction of a cross-shaped motif, or so-called 4 × 4 tile [four four-arm junctions linked together through flexible dT4 linkers (Figure 2c)], that was used to template the formation of conductive nanowires or protein 2D arrays (26). Mao’s group took advantage of the C4 symmetry of the 4 × 4 tile and introduced sequence symmetry into the DNA strands in the

FIGURE 2: Models of some representative DNA tiles and their assemblies into periodic 2D arrays. (a) Parallelogram DNA tile formed by joining four Holliday junctions in parallel. (b) Double-helix (DX) tile formed through strand exchange between two DNA duplexes. (c) A cross-shaped tile with four arms (4 × 4 tile). Each arm represents a four-arm junction. (d) Six-helix bundle tube tile viewed from the end of the tube. For panel a-d, representative AFM images of the 2D arrays are shown below the corresponding cartoon models. (e) DNA origami: (left) principle of DNA origami, folding long ssDNA into shapes by multiple helper strands; (middle) star and smiley face DNA origami tiles self-assembled by foldinga7k b ssDNA with more than 200 helper strands; and (right) hairpin loops (white dots) that can be introduced into certain helper strands to accurately display designated geometries on the fully addressable origami tiles.

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Downloaded by UNIV EST PAULISTA UNESP on July 24, 2009 Published on January 27, 2009 on | doi: 10.1021/bi802324w same tile. By doing this, they effectively minimized the numberof uniqueDNA strandsrequiredand reducedpossible experimental errors (27). They later applied the same principle to the design of other symmetric tiles such as threepoint and six-point star motifs (28, 29). Nicely formed 2D lattices spanning up to square millimeters were observed in these works, which can be attributed to reduced distortion conferredby the sequencesymmetry.In additionto the planar tiles mentioned above, 3D six-helix (Figure 2d) and threehelix DNA bundle tiles were built (30, 31). These tiles can form nanotubes with fixed diameters, periodic 2D lattices, and potentially 3D crystals.

An exciting breakthrough was made by Rothemund as he first presented “scaffolded DNA origami” (Figure 2e) which was formed by folding M13mp18 genomic DNA (7 kb) into desired shapes (∼100 nm in diameter) with the help of more than 200 short DNA strands (known as helper strands) (32). The versatility of the system was demonstrated by the formation of five arbitrary geometries, including rectangles, squares, triangles, stars, and smiley faces. DNA origami is a fully addressable molecular pegboard with more than 200 six-nanometer pixels because each helper strand at a specific position has a unique sequence. The addressability was demonstrated by introducing DNA hairpins into the helper strands at certain positions to display arbitrary characters (e.g., “DNA”) or shapes (e.g., diagram of DNA double helix) on the origami tile. Individual origami tiles can be further assembled into 2D arrays through base pairing between extended helper strands on one tile and the unpaired scaffold DNA (7 kb ssDNA) on another. “Scaffolded origami” selfassembly is a powerful tool for generating finite addressable nanostructures in both two and three dimensions. For example, Shih and colleagues constructed DNA octahedrons (3) and nanotubes (34) with fixed dimensions using either artificial or natural ssDNA as a scaffolding strand. More complicated patterns can be potentially accessed by selectively combining homogeneous or heterogeneous origami tiles through programmed connectivity, as will be discussed in the next section.

(Parte 1 de 4)