DNA Origami

Nanotechnological approaches toward cancer chemotherapy

Rukkumani Rajagopalan , Jatinder V. Yakhmi , in Nanostructures for Cancer Therapy, 2017

3.5 DNA Origami

DNA Origami is one of the most recent techniques of utilizing DNA as building blocks for synthesis of nanoparticles. It is one of the latest methods in the field of nanotechnology, having its own limitations and opportunities. "Origami" is a Japanese word that means folding of plain sheet into an arbitrary form having a specific dimension. Long strands of DNA are folded into a complex scaffold of staple strands having 200–300 nucleotides. This leads to formation of a complex structure that has characteristic features because of their nanoscale dimensions ( Tørring et al., 2011). These DNA nanostrustures are known to still be in their preliminary developmental stages, since key domains, such as their biocompatibility and physiochemical characterizations are yet to be established. However, theoretically, DNA origami has the immense potential to contribute significantly in a wide range of fields, such as diagnosis and drug delivery (Zhan et al., 2014). Cancer therapy and diagnosis is one such potential domain where DNA origami showed significant anticancer efficacy and may contribute immensely. Zhang et al. (2014) demonstrated that doxorubicin-loaded, triangle-shaped DNA origami could be an efficient and safe innovative platform for treating breast cancer in nude mice (Fig. 8.2).

Figure 8.2. Various Platforms of Nanostructures

(A) PEG-coated nanostructure, (B) aptamer-coated nanostructure, (C) peptide-coated nanostructure, (D) liposomal nanostructure, (E) polymeric nanostructure, and (F) dendrimer-based nanostructure.

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Nanotechnology Tools for the Study of RNA

Hisashi Tadakuma , ... Takuya Ueda , in Progress in Molecular Biology and Translational Science, 2016

2.1.3 Scaffold Design (DNA Origami)

DNA origami takes a much different approach from that of multistranded and SST design. 42 Usually, 7249-nucleotides long single-strand circular genomic DNA of M13mp18 phage is used as scaffold and hundreds of short helper strands called staples are used to fold longer scaffold into specific structure. In the folding process, scaffold DNA acts as a guide or a seed, 51 which increases the efficiency of folding and robustness to the stoichiometry of strands.

In 2006, "Smiley face" shook the DNA nanotechnology field (Fig. 3A). 42 Rothemund changed the rule of the game from assembling short strands motif into a large structure to fold long scaffold into specific structure. 52 The long scaffold of DNA origami is fold and hold by crossover made by staple strands. Typically, the staple strands bind to three adjacent helices, and the length is commonly 32 nucleotides, in which central 16 nucleotides bind to one helix and the remaining two parts of 8-nucleotide ends bind to the adjacent helices (Fig. 3B). The helical turn of DNA is usually approximated to be 3–3.5   nm in length and 3.5   nm in width. One helical turn (10.67 nucleotides) is different from that of canonical DNA (10.4 nucleotides/turn), resulting in the slightly twisted structure. Therefore, to relax the strain, usually one nucleotide is omitted every 48 nucleotides. 53 The length (about 3.5   nm) is also slightly different from that of canonical DNA (3.4   nm), which might be due to the interhelix gap presumably induced by electrostatic repulsion. Folded structures with straight edges sometimes stick together due to Ï€–Ï€ stacking. To prevent this aggregation, single-strand 4T hairpin loops (four thymidines) are introduced to the staple strands located at the edge and corner part. If the stacking of folded DNA origami cause severe problem, one can design the edge with concavity and convexity.

Figure 3. Scaffold design (DNA origami). (A) Smiley face structure with DNA origami method. (B) In DNA origami method, long circular single-stranded DNA (black) is folded into the desired shape by many short single-stranded DNAs (termed "Staple"), the latter typically bind to three adjacent helices, and the length is commonly 32 nucleotides, in which the central 16 nucleotides bind to one helix and the remaining two parts of 8-nucleotide ends bind to the adjacent helices. Unit pixel size is with dimensions of 3.6   ×   3.5   nm.

 Part A, B: adapted from Rothemund (2006), images reproduced with permission from Nature Publishing Group (NPG). 42

Folding of DNA is performed by adding a 5- to 10-fold excess of each staple strand, and by annealing the sample using PCR machine with ramp method (decrease the temperature of the sample with time) or at constant temperature. 54 Folded DNA origami can be purified by column (ultrafiltration, gel filtration), by gel electrophoresis, 55,56 or by PEG precipitation. 57,58 The yield of folding is quite high (90–95%) and the homogeneity is also high. 59

DNA origami also can be folded into 3D objects, 60,61 where the architecture is developed from a six-helix bundle (6HB) DNA nanotube. 62 In this architecture the unit length is 7 nucleotides and not 8 nucleotides. Seven nucleotides correspond exactly to 2/3 of a turn and 14 nucleotides correspond exactly to 4/3 of a turn, therefore, crossover between adjacent helix is allowed in honeycomb 6HB bundle structure, where six helices rotated 120 degrees to each other (Fig. 4A). These features allow connecting multiple honeycomb layers, enabling the formation of 3D objects (Fig. 4A). Further introduction of twist and curve, more complicated structure of gear, 63 box, 64–66 pot, 67 and sphere 68 were made (Fig. 4B). Recently, with the aid of graph theory and relaxation simulation, a general method of folding arbitrary polygonal digital meshes into the structure was reported, in which the design process is highly automated. 69 Thus, various types of structures could be made by scaffold design (DNA origami) methods.

Figure 4. 3D DNA structure of Scaffold design. (A) Basic unit of scaffold designed 3D DNA structure. Cross-section (left) and side view (right) of honeycomb structure composed of six helices with sample staples using caDNAno (http://cadnano.org/, bottom). 60,70 See also Section 5.3 for detailed design processes. (B) Representative 3D structures such as gear and pod.

 Part B: adapted from Deitz et al. (2009) (left) 63 and Han et al. (2011) (right). 67

Highly assembled structure of DNA origami is also possible. Using pole and joint approach Yin and coworkers made hexagonal prism (60   MDa, Fig. 5A). 71 With 100-nm edges, the sizes of these structures become comparable to those of bacterial microcompartments such as carboxysomes. The joint pole termed DNA "tripod" is a 5-MDa 3-arm-junction origami tile, in which interarm angles and pole (arm) length can be controlled, so that, with the connector sequence design, many types of structures such as a tetrahedron (–20   MDa), a triangular prism (–30   MDa), a cube (–40   MDa), a pentagonal prism (–50   MDa), and a hexagonal prism (–60   MDa) can be self-assembled. In a tripod, each arm has an equal length (–50   nm) and contains 16 parallel double-helices packed on a honeycomb lattice with twofold rotational symmetry, and "struts" consisting of two double-helices support and control the angle between the two arms. The yields of tripod-assembled structures are highly dependent on the number of vertexes: 45, 24, 20, 4.2, and 0.11% for the tetrahedron, the triangular prism, the cube, the pentagonal prism, and the hexagonal prism, respectively. Dietz and coworkers took another approach to make polymerized structures with dynamic structure change. 72 Inspired by the interaction between an RNA-based enzyme ribonuclease P (RNase P) which cleaves the 5' leader sequence for tRNA maturation and its substrate pretransfer RNA (tRNA). They used shape complementarity to assemble multiple DNA origami. In RNase P recognition, the acceptor stem and the TΨC loop of tRNA fit to the binding pocket of RNase P by a few nucleobase stacking interactions with the S domain of RNase P (Fig. 5B). 73 Similarly, in RNase P-inspired shape recognition method, blunt-ended double-helical DNA protrusions on one motif assume the role of the tRNA acceptor stem and corresponding concave on another motif mimic the RNase P binding pocket, and the nucleobase stacking bonds connect two motifs. 53,74–76 Upon two motifs engage, nucleobase stacking interactions occur at the double helical interface of the shape complementary protrusions and concave, but only when the helices fit correctly. Nucleobase stacking interaction method is sensitive to the concentration of counter ions, such as monovalent and divalent cations in the solution because repulsion between the negatively charged surfaces of DNA affects the equilibrium of the interaction, which allows on-off switching of the interface. All in all, without base pairing, RNase P-inspired method with nucleobase stacking bonds can build up micrometer-scale one- and two-stranded filaments and lattice, and transformable nanorobot. The merit of DNA origami is the design ability and robustness. As mentioned earlier, many types of structures have been made with high yield. In addition, long single-strand DNA scaffold can be a backbone, such that the structural rigidity to be ensured. 77 The limitation is based on the length of long scaffold. However, long scaffolds such as lambda DNA/M13 hybrid DNA scaffold (51,466 nucleotides), 78 PCR amplification-based scaffold [26   kb nucleotide fragment of lambda DNA (48,502   kb)], 79 and double strand form of lambda DNA itself 80 was used instead of M13mp18 scaffold (7,249 nucleotides). Combining these methods with higher order assemble method described previously, the limitation of scaffold may not be a problem from a practical point of view.

Figure 5. Higher assembled DNA structure. (A) Pole and joint approach to construct higher assembled DNA structure. A tripod is composed of one set of DNA origami structure and used as a basic unit that has three arms and binds with each other at the apical point. The structure of the end product is defined by the angle between the arms. (B) Shape-complementarity method to construct higher assembled DNA structure. (Top) DNA structure binds to its paired structure (top right) in a way that RNase P recognizes its substrate tRNA (top left). (Middle) Upon two motifs engagement, nucleobase stacking interactions occur at the double helical interface of the complementary shapes, but only upon correct fit of the helices. (Bottom) Shape-complementary interaction is highly affected by ion concentration, therefore higher assembled DNA structure, for example, nanorobot of 15 MDa, can be reversibly transformed in three different conformation states: disassembled, assembled with open arms, and assembled with closed arms, respectively, by changing the Mg2+ concentration.

 Part A: adapted from Iinuma et al. (2014). 71 Part B: adapted from Gerling et al. (2015). 72

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Quantitative Imaging in Cell Biology

Susanne Beater , ... Philip Tinnefeld , in Methods in Cell Biology, 2014

24.2 Functionalizing DNA Origami Structures

The DNA origami structures can be viewed as molecular breadboards. As each of the short staple strands can be extended and functionalized in many different ways, these constructs do allow not only specific binding on surfaces but also dye labeling in defined distances with nanometer accuracy ( Stein, Schuller, Bohm, Tinnefeld, & Liedl, 2011; Steinhauer et al., 2009). Above that, a high degree of control is maintained over the number of fluorescent dyes. In another variation, the binding of proteins and other nanoobjects can be realized by functionalizing them with DNA and binding them via the complementary sequence to the DNA origami (Andersen et al., 2009; Shen, Zhong, Neff, & Norton, 2009). Modified DNA origamis can be either used to study bioassays on the surface without changing their behavior compared to an ensemble experiment (Gietl, Holzmeister, Grohmann, & Tinnefeld, 2012) or used as fluorescence and superresolution standards (Schmied et al., 2013, 2012; Steinhauer et al., 2009).

Different methods of functionalizing or labeling the DNA origami constructs have been established. The most straightforward approach depicted in Fig. 24.2 is to functionalize one or more of the staple strands before the whole folding process. This leads to the so-called internal labeling of the structure. This method is very robust and gives very high yields of correctly labeled DNA origamis. Its obvious disadvantage is that only temperature-insensitive modifications can be folded with this method. As the folding process occurs at temperatures starting over 90   °C, the functionalization has to be stable enough to survive this process. The method of internal labeling is also not very cost-efficient especially when several identical modifications are intended: each of the staples that one wants to modify has a unique sequence that needs to be functionalized.

Figure 24.2. Labeling DNA origami. Internal labeling of DNA origami structures is accomplished by the substitution of a staple strand by its functionalized version, for example, a dye-labeled DNA strand.

Another labeling approach is external labeling, which is illustrated in Fig. 24.3. For external labeling, the staple strands one wants to modify are not directly functionalized but prolonged by a specific DNA sequence of about 20 bases. This extended sequence then protrudes from the folded DNA origami and works as docking strand. This docking strand binds a complementary sequence with the desired modification. The external labeling can either be carried out in one step, which means that the whole construct (scaffold strand, unmodified and extended staple strands, and modified complementary sequence) is folded at once. Also, subsequent labeling is possible, which means that first, the DNA origami with the extended docking strands is folded and purified and then the functionalized counter strand is added, incubated for a short time (typically 2   h; Lin et al., 2012) and purified. The subsequent labeling can be done at milder conditions, especially at much lower temperatures (37   °C is common; Lin et al., 2012). Additionally, by using external labeling, a high number of identical modifications in one DNA origami become more cost-efficient. As the individual staple strands do not have to be expensively modified but only extended and not more than one modified counter strand is required, the costs decrease drastically. Using subsequent labeling, parallelization of the fabrication of differently labeled DNA origamis becomes easier as the basic construct remains unchanged and only the desired modified counter sequence has to be added after purification. Generally, however, external labeling goes along with lower yields compared to internal labeling.

Figure 24.3. External labeling of DNA origami structures is realized by extending specific staple strands by about 20 bases that protrude from the folded structure and act as docking strands for the functionalized counter strand that can be added either before folding or after folding and purification. The modification of the counter strand can be pointing either toward the DNA origami or away from it. The latter causes less steric hindrance but is less accurate in terms of the exact position of the modification.

One more way of modifying the DNA origami is the so-called enzymatic labeling (Jahn et al., 2011). This technique is practically a composition of external and internal labeling: The individual staple strands one wants to modify are enzymatically labeled with either a fluorophore or other groups, namely, biotin, amine, and digoxigenin groups. The modified staples are then simply mixed with the unmodified staple strands, scaffold, and buffer and annealed as usual.

The enzyme used in this reaction is DNA nucleotidylexotransferase that attaches dideoxynucleotide triphosphates to the 3′ end of the staple strands. By modifying the dideoxynucleotide triphosphates with a fluorophore or another modification as listed earlier in the text, the staple strand is extended by a labeled nucleotide. Dideoxynucleotides also ensure that only one single-labeled nucleotide is attached as the reaction stops after the integration. This makes the labeling very controllable. As this enzymatic labeling can be done for several staple strands at once, it provides a cost-efficient way of labeling with certain functionalities.

Specific binding of DNA origamis to surfaces is mostly accomplished using biotin-modified DNA origami structures (about five biotin anchors per structure are common) and via an avidin derivate (NeutrAvidin or streptavidin) onto a BSA/biotin-coated surface. This method leads to very little unspecific background caused by excess staple strands or free dye. In contrast to electrostatic binding (which occurs on, e.g., poly-l-lysine surfaces), the DNA origami only binds in a predetermined orientation. This enables structures such as DNA origami nanopillars to stand upright (Schmied et al., 2013). Besides biotin and fluorescent dyes, also fluorescent proteins can be used to label DNA origamis as long as they can be functionalized with DNA (Shen et al., 2009).

The labeling density (which means the number of modifications per area) is mainly limited by steric constraints. In principal, each staple strand could be modified several folds, but at some point, the folding will be inhibited. Also, self-quenching of the fluorescent dyes can occur when the dyes get close enough to affect each other. It has been shown that modification of every staple at one end (3′ or 5′) is possible without steric hindrance or self-quenching of the fluorescent dyes (Schmied et al., 2012). Especially, bundle-like structures are sufficiently rigid and offer a high labeling density. This can be exploited for quantitative analysis: Samples with different but defined numbers of fluorescent dyes can act as brightness references. Also, defined distances between two marks of fluorescent dyes can be used for quantifying the resolving power of a microscope. As for high-resolution methods like STED and SIM, a high density of dyes is beneficial, as DNA origamis qualify as STED or SIM rulers.

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Metabolons and Supramolecular Enzyme Assemblies

Eiji Nakata , ... Takashi Morii , in Methods in Enzymology, 2019

3.3.4 Notes

1.

DNA origami buffer with various pH ranging from 7.0 to 9.0 showed little or no influence for the loading yield of POI-fused adaptors on DNA scaffold. DTT and zinc ion are crucial for maintaining the activity of modular adaptors containing the zinc finger domain.

2.

By using modular adaptors, the assembly on DNA scaffold could be kept at ambient temperature for 24   h without significantly reducing the assembly yield.

3.

For orthogonal assembly of three POIs at defined positions on DNA scaffold, one-pot reaction is possible due to the high orthogonality and high reactivity of the optimal set of orthogonal modular adaptors. Each 100   nM of three adaptor-fused POIs were added to 5   nM DNA scaffold at the same time. After 30   min incubation on ice or 10   min at ambient temperature, the POIs were simultaneously assembled at the designed positions with high yield and orthogonality.

4.

The yield of ZS-XR on DNA scaffold (P specific) was calculated as the percentage of the number of modified DNA scaffolds bearing ZS-XR at the expected position (N expected posi) over the total number of well-formed DNA scaffold (N total):

P specific  =   (N expected posi/N total)   ×   100.

The yield of ZS-XR located at unexpected positions (P nonspecific) was calculated as the percentage of cavities modified nonspecifically by ZS-XR (N unexpected posi) over the total number of cavities of well-formed DNA scaffold (2N total):

P nonspecific  =   (N unexpected posi/2N total)   ×   100.

Typical examples are found in our previous reports (Kurokawa et al., 2018; Nakata et al., 2015, 2012, Ngo et al., 2014, 2016; Nguyen et al., 2017).

5.

The yield of coassembled ZS-XR, AC-XK and AH-XK on DNA scaffold (P coassembly) was calculated as the percentage of the number of modified DNA scaffolds bearing the three enzymes (ZS-XR, AC-XK and AH-XK) at the expected positions on the DNA scaffold (N expected posi) over the total number of well-formed DNA scaffold (N total):

P coassembly  =   (N expected posi/N total)   ×   100.

Typical examples are found in our previous reports (Ngo et al., 2016; Nguyen et al., 2017).

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Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods

Yihong Feng , ... Masayuki Endo , in Methods in Enzymology, 2020

5 Notes

1.

The DNA origami nanocapsule structure was designed using the caDNAno software ( http://cadnano.org/) (Douglas et al., 2009b).

2.

Details on the information of the instrument are available at the homepage of RIBM (http://www.ribm.co.jp).

3.

For HS-AFM imaging, small cantilevers are used. Small cantilevers (9   μm long, 2   μm wide and 130   nm thick; BL-AC10DS, Olympus, Tokyo, Japan) made of silicon nitride with a spring constant ~   0.1   N/m, and a resonant frequency of ~   300–600   kHz in water are commercially available from Olympus.

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Nanobiotechnology

David P. Clark , Nanette J. Pazdernik , in Biotechnology (Second Edition), 2016

DNA Origami

Building nanostructures by assembling multiple different DNA molecules becomes extremely difficult beyond a certain level of complexity. The DNA origami approach greatly simplifies building DNA nanostructures by using one very long DNA strand and folding it up to form a scaffold. A number of much shorter "staple strands" are added in excess to help folding. The "staple strands" bind at specific sites along the longer scaffold strand to drive folding ( Fig. 7.21). This approach means that it is no longer necessary to strictly control the ratio of different DNA strands as for the "traditional" DNA folding described in the previous section (see especially Fig. 7.19). Assembly is much faster and yields are much higher with the origami approach.

FIGURE 7.21. Principle of DNA Origami

(A) The traditional approach uses multiple strands to build DNA nanostructures. (B) DNA origami uses one long scaffold strand plus several short staple strands that guide folding.

 From Rothemund PWK (2005). Design of DNA origami. Proc Int Conf Computer-Aided Design (ICCAD) 471–479; figure provided by Paul Rothemund, Computation and Neural Systems, Caltech.

Except in very simple cases, DNA origami relies on computer-aided design, in particular for specifying the DNA sequences required for building the chosen shapes. Figure 7.22 illustrates the procedure for building a complex structure by using this approach. The traditional approach for such structures would take weeks and involve synthesizing and purifying multiple long strands. These must then be assembled in the correct proportions and in the correct order. DNA origami, in contrast, requires one scaffold strand plus a roughly 10-fold excess of the staple strands. These strands are all mixed together, heated, and then cooled slowly to anneal. This process takes only a few hours.

FIGURE 7.22. Steps in Designing DNA Origami

(A) Fill the chosen shape with helixes plus crossovers (needed for stability). (B) Convert design to a single long folded scaffold. (C) Insert staple strands to bind scaffold into shape. (D) Helical representation.

 From Rothemund PWK (2005). Design of DNA origami. Proc Int Conf Computer-Aided Design (ICCAD) 471–479; figure provided by Paul Rothemund, Computation and Neural Systems, Caltech.

In DNA origami, nanostructures are built by folding up a single very long strand of DNA. Many much smaller staple strands assist in the folding.

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Reconstituting the Cytoskeleton

Brian S. Goodman , Samara L. Reck-Peterson , in Methods in Enzymology, 2014

2.3 Determining the integrity of the folded structure

To determine the integrity of the folded DNA origami structure, several methods can be used. Here we briefly describe agarose gel electrophoresis, transmission electron microscopy (TEM), and DNA PAINT.

1.

Agarose gel electrophoresis can be used to visualize the folded chassis compared to the initial input in the folding reaction. The folded chassis will run faster in the gel. Run the folded chassis on a 2% agarose, 1   × TBE gel containing 11   mM MgCl2, and 0.7   μg/ml ethidium bromide. The MgCl2 should be added after boiling the agarose in TBE buffer. Run the gel at a constant voltage of 70   V for 90   min (Fig. 10.2B). Running the gel at a voltage >   70   V will cause the gel to overheat, causing the DNA origami structure to denature and run as a smeared band. We routinely place our gel apparatus in an icebox to dissipate heat and exchange the running buffer if the gel is run for longer than 2   h.

2.

TEM techniques can be used to assess the structure using previously described methods (Douglas, Marblestone, et al., 2009).

3.

DNA PAINT techniques can be used to observe handle incorporation (Fig. 10.2C) as described previously (Derr et al., 2012; Jungmann et al., 2010). To immobilize the DNA origami on a cover slip, biotin-labeled handles are included during the folding reaction. The biotin-labeled origami structures can then be adhered to avidin-coated cover slips. Previously, using the DNA PAINT method we found that handles are incorporated with 80% efficiency. Modifying staple length and other design principles may improve the efficiency of handle incorporation (Sobczak et al., 2012).

Solutions

1.

10   × TBE buffer

•

450   mM Tris, pH   8.1

•

450   mM Boric acid

•

10   mM EDTA

2.

Agarose gel

•

2% Agarose

•

1   × TBE buffer

•

Boil to dissolve agarose

•

Add MgCl2 to 11   mM

3.

Gradient buffers

•

1   × TBE buffer

•

11   mM MgCl2

•

15–45% glycerol

4.

Agarose gel running buffer

•

1   × TBE buffer

•

11   mM MgCl2

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Self-Assembled Nanostructures (SANs)

Mina Mekhail , ... Maryam Tabrizian , in Biology and Engineering of Stem Cell Niches, 2017

8.4 DNA Origami

Controlling the monomeric sequence allowed polymer chemists to design the physicochemical properties of synthesized nanoparticles. Similarly, controlling the nucleotide sequence in DNA and taking advantage of the Watson-Crick base pairing phenomenon, allowed molecular biologists to create DNA-based nanoparticles with any shape or size. 118 Nadrian C. Seeman, 119 was first to propose using DNA as building blocks to create immobile junctions, which can then be arranged to form more complex 3D structures. The field of DNA nanotechnology has since exploded into existence, with investigators designing a myriad of DNA nanostructures, both 2D and 3D, that can be used in a range of applications from microelectronics to drug delivery. 120,121 In this chapter, we will focus our attention on DNA origami and its application in the field of drug delivery.

"Scaffolded DNA origami," a term coined by Paul Rothemund, 118 is a method of folding a long single strand of DNA (scaffold) into precise shapes by using multiple short oligonucleotide "staple strands" to stabilize the overall structure (Fig. 25.5A). First, the desired shape is drawn and filled with the scaffold strand in a raster fill arrangement. A computer program is then used to design and place the staple strands on the scaffold strand to fold it into the desired shape. The scaffold and staple strands are mixed, heated, and then cooled down to allow the self-assembly of the DNA origami. 118 This precise control over DNA nanoparticle fabrication prompted many researchers to apply DNA origami for drug delivery applications.

Figure 25.5. (A) Scaffolded DNA origami, a method that involves the use of a long single strand of DNA and shorter DNA sequences (staples) to form virtually any 2D or 3D shape. The images on top are the computer-produced folding sequence of the DNA strand and the bottom row is an atomic force microscopy image of the different DNA origami shapes; (B) It has been shown that different shapes can illicit different biological effects. The triangular-shaped origami provided the best accumulation at the breast tumor site as compared to square and tube-shaped origami. Doxorubicin was intercalated within the DNA and was readily released at the tumor site due to low pH; (C) (i) A barrel-shaped nanorobot used as a drug delivery system. The nanorobot has covalently stabilized hinges in the back, and an aptamer lock mechanism in the front. The aptamers dissociate in the presence of an antigen and releases the payload. (ii) TEM images of the nanorobot in closed and open configurations.

 Adapted from (A) Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature 2006;440(7082):297–302; (B) Zhang Q, Jiang Q, Li N, Dai LR, Liu Q, Song LL, et al. DNA origami as an in vivo drug delivery vehicle for cancer therapy. ACS Nano 2014;8(7):6633–43; and (C) Douglas SM, Bachelet I, Church GM. A logic-gated nanorobot for targeted transport of molecular payloads. Science 2012;335(6070):831–4.

In one study, it was shown that triangular DNA origami with side lengths of 120   nm was able to accumulate much more readily at the tumor site due to passive targeting as opposed to square and tube-like DNA origami (Fig. 25.5B). 122 Moreover, the triangular DNA origami was able to retain (via intercalation) and release doxorubicin at the tumor site, and caused no systemic toxicity. 122 Triangular and tubular DNA nanostructures have also been shown to effectively enhance doxorubicin uptake in doxorubicin-resistant MCF-7   cells. 123 In another, more complex structure, a DNA origami nanorobot was designed for targeted drug delivery. 124 The nanorobot, which resembles a hexagonal container with a lid, has an aptamer-based locking mechanism that interacts with target proteins to open and release the payload. The investigators fabricated one of those nanorobots (35   ×   35   ×   45   nm) using 196 oligonucleotide staple strands and a 7308-base scaffold strand (Fig. 25.5C). 124 There are more examples demonstrating the effectiveness of using DNA origami nanostructures in drug delivery applications, and the readers are referred to an excellent review on this subject matter. 121

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Transforming Synthetic Biology with Cell-Free Systems

Arnaz Ranji , ... Michael C. Jewett , in Synthetic Biology, 2013

DNA Origami

The field of structural DNA nanotechnology was born in the 1980s with initial work by Seeman, who proposed building geometric structures with multiple strands of DNA at a nanoscale level. 1,55 However, complications in synthesis and stoichiometric control left the field lacking in more promising developments. 1 In 2004 the Joyce group demonstrated that a long single-stranded DNA could be folded into a geometric shape (octahedron) with the help of synthetic shorter oligomers. 56 Taking this further, Paul Rothemund, in a landmark discovery, demonstrated that long single-stranded DNA could be folded at nanoscale levels with the help of smaller staple strands to create various shapes. 57 Specifically, Rothermund demonstrated the creation of complex single-layered nanostructures that were 100   nm in diameter and had a spatial resolution of 6   nm. 57 Termed DNA origami, this approach creates custom nanoscale shapes that are atomically precise by taking advantage of the specificity of Watson Crick base-pairing.

In recent efforts, there has been a thrust to make 3D DNA origami structures and expand the dimensionality and functionality of molecularly engineered objects. Douglas et al., for example, demonstrated the ability to make a variety of 3D shapes from linear DNA molecules. 58 However, proper assembly of the 3D structure required week-long folding times and was much more complicated than 2D origami. 58 New technology by the Yan group has created a more sophisticated version of DNA origami termed DNA kirigami that involves folding and cutting DNA into topological objects. 59 Additionally, the same group created a strategy to engineer 3D DNA structures with complex curvature. 60 As the fundamental design principles of complicated architectures become more and more elucidated, including computer-aided design caDNAno, 61 this field is now turning to applications. 1 Key among these is the creation of DNA nanochips that can be used to observe single molecule behavior of DNA-binding enzymes, 62 and DNA nanorobots for medical therapeutics and medical diagnostics. 63 In particular, the recent demonstration that DNA nanorobots can target cancer cells and deliver an antibody payload 63 is expected to usher in a new era of DNA device utilization.

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Nanotechnology Tools for the Study of RNA

Hirohisa Ohno , Hirohide Saito , in Progress in Molecular Biology and Translational Science, 2016

1.1 RNA as a Nanomaterial

Biomolecules are often used as materials in the nanotechnology field. In particular, DNA has been widely investigated in expanding the field because of its molecular recognition and self-organization ability based on complementary Watson–Crick base pairing. DNA nanotechnology pioneered by Seeman 1,2 has been accelerated by the following report of DNA origami by Rothemund. 3 DNA origami technology enables us to design and construct a variety of two-dimensional (2D) and three-dimensional (3D) nano-scale structures (nanostructures) made of only DNA with high accuracy and efficiency. 4 Recently, in addition to DNA, bioengineers have also been attracted to the use of RNA. 5,6 In a similar manner to DNA molecules, RNA is also able to form Watson–Crick base pairing, and regulate gene expression through DNA–RNA and RNA–RNA interactions. However, the role of RNA is not limited to simple nucleic acid interaction. For example, transfer RNA (tRNA) molecules attached to a corresponding amino acid at their3'-terminus (reaction catalyzed by aminoacyl-tRNA synthetases), and ribosomal RNA plays central roles to catalyze peptide-bond formation within ribosome complex. Furthermore, discovery of various RNA molecules such as ribozymes and riboswitches supports important and versatile functions of RNA in living systems. Why can RNA molecules play variety of functions? Structural analysis of RNA revealed that functional diversity of RNAs is based on their complex 3D structures like proteins. Importantly, these complex RNA structures are made by the self-assembly of many smaller and unorthodox interactions between base–base, base–sugar and base–phosphate. Such distinctive interactive regions with structures are called RNA structural motifs (RNA motifs). So far, a variety of RNA motifs have been found in nature and extracted as building blocks (Fig. 1) to construct artificial nanostructures. 7

Figure 1. RNA structural motifs. Various RNA structural motifs have been found out from natural RNA molecules. Each 3D data was obtained from PDB (id is shown later). right-angle (RA) motif (extracted from 1JJ2 8 ); 180° kissing-loop (KL) motif (1JJM 9 ); three-way junction (3WJ) motif (extracted from 4V4Q 10 ); tRNA motif (extracted from 4TNA 11 ); IIa RA motif (2NOK 12 ); pRNA motif (3R4F 13 ); 120° KL motif (2BJ2 14 ); loop-receptor motif (extracted from 1HR2 15 ). Core region of each motif is colored and white region corresponds to normal Watson–Crick type double-stranded helix region. All of them are drawn in the same scale.

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