Focus: Small Science DNA FUHGLW&KHOVHD*RUGRQ+65 Nano %\$PULWD*R\DO technology ith these famous words, James WWatson and Francis Crick ush- stranded DNA and the energetics of ered in a new era in biology—the age DNA folding in solution are highly pre- of DNA. Just as the implications of GLFWDEOH  6SHFLÀF'1$VHTXHQFHV DNA’s complementary base pairing of hundreds of base pairs can be easily and helical structure for a possible synthesized in the laboratory. DNA is mechanism of replication did not es- also extremely easy to replicate, either “We wish to suggest a cape them, the possible applications by the polymerase chain reaction (PCR) structure for the salt of DNA to materials science, based on or by bacterial plasmid methods. the same elements of DNA’s structure, Molecular engineers are thus in pos- of deoxyribose nucleic have not escaped the notice of present- session of two of the three require- acid (DNA). This struc- day chemists. DNA is the next frontier ments for constructing nanostruc- of nanotechnology and will become tures—a well understood material with ture has novel features the basis of the construction of many which to build, and methods by which which are of consider- three-dimensional nanostructures. to produce and manipulate the mate- able biological inter- DNA is the ideal macromolecule to ULDO7KHÀQDOLQJUHGLHQWQHFHVVDU\LVD utilize in the quest to develop self-as- versatile design strategy (3). Over the est… It has not escaped sembling nanostructures. Molecular last ten years, DNA nanostructure tech- our notice that the spe- self-assembly—the synthetic strategy nology has gone through three distinct FLÀF>FRPSOHPHQWDU\ of choice for scientists developing new phases. Dr. Nadrian Seeman of New nanotechnologies—is the spontaneous,

occurring DNA structures: sticky ends and branched DNA (5). Sticky ends are short, single-stranded regions of DNA that extend from the end of a double-stranded DNA mol- ecule. Sticky ends are able to act like 9HOFURÁDSVE\YLUWXHRI '1$·VLQKHU- ent ability to engage in complementary 421 (2003): 427-31. base pairing; they can come together to form normal, double helical DNA Nature of predictable geometry. However, al- FUHGLW$GDSWHGIURP6HHPDQ1´'1$LQDPDWHULDO ZRUOGµ though they are very useful, sticky ends Figure 2. An illustration of the possible applications of DNA scaffolds. (a) Proteins are attached DUHQRWVXIÀFLHQWWRHQDEOHVFLHQWLVWV WRDVFDIIROGIRUWKHSXUSRVHRI;UD\FU\VWDOORJUDSK\ E ThHGHSRVLWLonRIPHWDOLRQVRQ DNA scaffolds at a key step in the synthesis of nanoelectronic materials (5). to construct highly interesting shapes. Sticky ends are invaluable in self-as- Holliday junctions have branch points the structures, which would facilitate sembly, and allow the construction of ÁDQNHGE\WKUHHWRHLJKWDUPVRI '1$ very precise synthesis (Figure 2) (5). circular DNAs and extended linear  7KHVHDUWLÀFLDOFRPSOH[HVSURYLGH However, as useful as the tile method sequences, but are limited in terms of QDQRPDWHULDOVHQJLQHHUVZLWKWKHÁH[- is in synthesizing nanocrystals, it has expanding the possible geometries of ibility to construct shapes that range distinct limitations. Because of the DNA nanostructures (5). from cubes to truncated octahedrons topology of the requisite branched Although DNA is unbranched and to Borromean rings (6). structure, it is not possible to reproduce linear the vast majority of the time, dur- Armed with these tools, the synthesis the DNA using regular DNA poly- ing the crossing-over step of meiosis, a of a nanostructure such as a cube is merase, the enzyme that synthesizes process responsible for generating sex relatively simple. In his experiments, DNA strands in nature. In 2004, Dr. cells, DNA takes on a branched con- Seeman created four-branched DNA William Shih of Harvard University formation at short-lived sites known junctions, and from each double heli- achieved the mammoth task of suc- as Holliday junctions. Naturally occur- cal branch extended a sticky end. Large cessfully synthesizing a nanostructure ring Holliday junctions consist of four numbers of these junctions were then from a single strand of DNA that was strands of double helical DNA bound able to assemble into two-dimensional replicable by PCR (7). together at a branch point (Figure 1). crystals that consisted of interconnect- Synthetic DNA complexes based on ed rings of DNA. These crystals could DNA Gets a Bit Cagey then be ligated to form a three-dimen- Shih’s triumph of molecular engineer- sional cube that was several nanometers ing came in the shape of an octahedron. across (Figure 1) (5). This octahedron was constructed from

Nature Thus, the prescription for two-dimen- a single, 1700 base pair-long strand of sional DNA nanostructure synthesis DNA, the folding of which was driven according to Seeman’s tile method is by a denaturation-renaturation reaction, a straightforward one—synthesize in conjunction with the addition of branched DNA with programmed ÀYHQXFOHRWLGHORQJ'1$VWUDQGV sticky ends, and allow it to assemble that served to guide its folding process into a closed object or crystalline array. (7). Such crystalline scaffolds could be used There are several things that make as platforms to hold other molecules Shih’s strategy for synthesizing the octa- for the purpose of X-ray crystallogra- hedron quite notable. First, it is possible phy, or to create complexes of proteins. to replicate the 1700 base pair DNA Three-dimensional lattices could be strand using lab techniques as simple used as scaffolds for assembling other as PCR. Second, no covalent bonds FUHGLW$GDSWHGIURP6HHPDQ1´'1$LQDPDWHULDOZRUOGµ 421 (2003): 427-31. materials. For example, metal atoms were either made or broken during Figure 1. 2Q WKH WRS OHIW LV D GLDJUDP RI D +ROOLGD\MXQFWLRQ7KHSRUWLRQVPDUNHG++· could be incorporated into the struc- the assembly of the octahedron. Most 9DQG9·DUHFRPSOHPHQWDU\VWLFN\HQGVDQG ture to make a nanoelectric assembly, importantly, however, Shih’s method they adhere to one another as shown in the which could function as a circuit. DNA introduced the concept of using short WRSULJKWÀJXUH7KHVHWZRGLPHQVLRQDOFU\VWDOV FDQWKHQEHOLJDWHGWRIRUPWKUHHGLPHQVLRQDO binding enzymes could even be used to DNA strands to direct the folding of cubes. (5) direct or restrict metal deposition onto a longer strand. However, this process

spring 2006 • Harvard Science Review 35 Focus: Small Science

UHSUHVHQWVDVLJQLÀFDQWFKDOOHQJHLQWKDW shape of its faces (7). Numerous struc- RWKHUPDWHULDOVDWVSHFLÀFORFDWLRQVLQ the components must be mixed in their tures in nature also take advantage of the structure; it would even be possible H[DFWVWRLFKLRPHWULFUDWLRVIRUHIÀFLHQW the strength of this geometry, includ- to encode binding sites for the attach- assembly to occur (7). ing the protein coat of several viruses, ment of sequence-specific binding The octahedral geometry of Shih’s which is icosahedral in shape. proteins along the three dimensional nanocrystal is extremely important. Each octahedron has a diameter of framework (7). Most importantly for Each of the twelve edges of the oc- approximately 20 nanometers, which is synthesis of rigid supernanostruc- tahedron is made of double stranded equivalent to the size of a small virus. tures, a tetrahedral nanocrystal has helical DNA. These helical struts frame The cavities of these octahedrons are been synthesized as well (8). Together the eight equilateral, triangular faces. sized to accommodate a sphere with with its octahedral counterpart, such a Many of the other shapes that have a diameter of about 14 nanometers, structure could eventually be used as a been synthesized, including the cube which would make the octahedrons building block in the synthesis of larger and truncated octahedron, are subject useful for such applications as the structures held together with linkers to deformation by even moderate transport of proteins and other macro- (specially designed strands of single amounts of force. The octahedron, on molecules, and possibly even for drug stranded DNA) (9). the other hand, very strongly resists delivery. Complexes of these nano- The DNA cages might also be inte- deformation because of the triangular structures could be used to position grated with biological macromolecules 440 (2006): 297-302. Nature DSHVDQGSDWWHUQVµ FUHGLW5RWKHPXQG3:.´)ROGLQJ'1$WRFUHDWHQDQRVFDOHVK Figure 3.$QLOOXVWUDWLRQRIWKH'1$oULJDPLmHWKRG  .

36 Harvard Science Review • spring 2006 Focus: Small Science and used as carriers for other molecules, “bottom-up” fashion at the nanoscale including proteins and drugs. Addi- OHYHOKDVEHFRPHVLJQLÀFDQWO\PRUH tional chemical capabilities could be feasible with the development of DNA added to the structures by conjugating origami (10). One of the goals of nano-

them to aptamers, which are folded, 440 (2006): 297-302. structure synthesis through patterned single-stranded lengths of DNA that DNA origami is the development of ELQGVSHFLÀFDOO\WRVPDOOPROHFXOHVRU Nature a nanobreadboard, or a very small to ribozymes, or RNA molecules with circuit to which proteins, aptamers, catalytic properties (5). ÁXRURSKRUHVQDQRWXEHVQDQRZLUHV gold nanoparticles or any number of

DNA Origami DSHVDQGSDWWHUQVµ other molecules could be added. These Stars, smiley faces, rectangles, triangles, might ultimately be used to model com- and squares—this seemingly random plex protein assemblies and molecular assortment of shapes represents a electronic circuits (10). handful of the two-dimensional nano- DNA nanotechnology has applica- structures that Dr. Paul Rothemund of tions as disparate as X-ray crystallogra- the California Institute of Technology phy, protein array assembly, self-assem- has been able to synthesize out of DNA bling nanomachinery, nanoelectronics, using a self-assembly technique he has and nanocircuitry. The structure of called “DNA origami” (10). DNA was initially resolved through a Rothemund’s new synthetic strategy combination of chemistry, physics, and shatters many of the rules governing the insight of many brilliant scientists. nanostructure assembly that have been Now, scientists are using their knowl-

HVWDEOLVKHGLQWKHÀHOGRI '1$QDQR- FUHGLW5RWKHPXQG3:.´)ROGLQJ'1$WRFUHDWHQDQRVFDOHVK edge of the structure and characteristics technology. His method uses a few hun- Figure 4.3LFWXUHVRIVRPHRIWKHQDQRVWUXF- of DNA, in combination with their dred 16 to 32 nucleotide-long single- WXUHVV\QWKHVL]HdE\5RWKHPXQG  . intuition about the potential of this stranded DNA molecules to “staple” a strains (10). biological polymer, to revolutionize very long single strand into any desired The variety of shapes that can be materials science, chemistry, biology, two-dimensional shape. Individual synthesized using this method is limited and computing. only by the imagination. The synthesis staples can be made into nanometer- —Amrita Goyal ’09 is a Chemistry scale pixels that create surface patterns SURFHVVLVDOVRUHPDUNDEO\HIÀFLHQW)RU concentrator in Canaday Hall. on a given 100 nanometer shape or can any given shape, over 70% of the nano- combine shapes into larger structures. structures synthesized come out “well This process is capable of producing formed”(10). Moreover, the staples that nanostructures that are larger and more hold the scaffold together provide a References complex than any of those generated means of generating patterns of pixels, :DWVRQ-'DQG&ULFN)+´0ROHFXODUVWUXFWXUH by its predecessors (10). as they can be labeled with fluoro- of nucleic acids: a structure for deoxyribose nucleic Rothemund’s method is a surpris- phores, or small molecules that give off DFLGµNature 171 (1953): 737-8. :KLWHVLGHV*00DWKLDV-36HWR&7´0R- ingly straightforward one and is largely light when irradiated with a particular OHFXODUVHOIDVVHPEO\DQGQDQRFKHPLVWU\DFKHPLFDO computer-based. First, a shape is cho- frequency. Each oligonucleotide can VWUDWHJ\IRUWKHV\QWKHVLVRIQDQRVWUXFWXUHVµScience 254 (1991): 1312-9. sen, and on the computer, a model of serve as a six-nanometer pixel and be 6PLWK/0´7KHPDQLIROGIDFHVRI'1$µNature LWLVÀOOHGIURPWRSWRERWWRPZLWKDQ used to pattern complex designs on the 440 (2006): 283-4. 6HHPDQ1´7KHXVHRIEUDQFKHG'1$IRUQDQRVFDOH even number of parallel double helices. nanostructures. Examples of patterns IDEULFDWLRQµNanotechnology 2 (1991): 149-59.  6HHPDQ 1 ´'1$ LQ D PDWHULDO ZRUOGµ Nature Then, the single long scaffold strand of created by Rothemund include a picture 421 (2003): 427-31. DNA is folded back along the double of the DNA double helix accompanied *RKR$´6QDSS\'1$ORQJVWUDQGIROGVLQWRRF- WDKHGURQµScience News 165.7 (2004): 99. helices, and periodic crossovers are E\WKHOHWWHUV´'1$µVQRZÁDNHVDQGD 6KLK:4XLVSH--R\FH*-´$NLOREDVH introduced to provide rigidity. Staple map of the world (Figure 4) (10). single-stranded DNA that folds into a nanoscale oc- WDKHGURQµNature 427 (2004): 618-21. strands with a length of 16 to 32 nu- Although these nanostructures are :HLVV3´,QVWDQWQDQREORFNVRQHVWHSSURFHVV cleotides are added to hold the scaffold all two-dimensional, the extension of PDNHV WULOOLRQV RI '1$ S\UDPLGVµ Science News 168.24 (2005): 372. together in the desired shape by means this system from two to three dimen- *RRGPDQ53et al.´5DSLGFKLUDODVVHPEO\RI VLRQVVKRXOGQRWEHGLIÀFXOWJLYHQWKH ULJLG'1$EXLOGLQJEORFNVIRUPROHFXODUQDQRIDEULFD- of complementary binding (Figure 3). WLRQµScience 310 (2005): 1661-5. Finally, the structure is optimized on the established precedents. The design and  5RWKHPXQG 3 :. ´)ROGLQJ '1$ WR FUHDWH QDQRVFDOHVKDSHVDQGSDWWHUQVµNature 440 (2006): computer to minimize any destabilizing synthesis of functional materials in a 297-302.

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