DNA nanotechnology from the test tube to the cell

748 NATURE NANOTECHNOLOGY | VOL 10 | SEPTEMBER 2015 | www.nature.com/naturenanotechnology

DNA nanotechnology is a purists approach to biomolecular engineering. The field aims to create molecular structures and devices through the exclusive use of DNA as an engi-neering material. The well-characterized nature of DNA base-pairing provides an easy means to control DNA interactions; this sequence programmability has allowed the rational design of pre-cisely defined structures ranging in size from nanometres to milli-metres, and of molecular motors or circuits that can autonomously move or process information. There is currently no other molecular engineering technology that enables the fully denovo design of a similarly complex and diverse set of biomolecular systems.

The success of DNA nanotechnology comes from three key ingredients: 1) our quantitative understanding of DNA thermo-dynamics, which makes it possible to predict reliably how single-stranded DNA molecules fold and interact with one another1,2; 2) the rapidly falling cost and increasing quality of DNA synthe-sis3; and 3) the focus on cell-free settings, where designed reaction pathways can proceed without interference from DNA and RNA processing enzymes and other confounding factors that might be encountered in cells.

DNA nanotechnology has long been motivated by the goal of building smart therapeutics, drug delivery systems, tools for molec-ular biology and other devices that could interact with or operate within living cells47 (Fig.1). Such applications play to the obvious strengths of nucleic acid nanostructures and devices, particularly their small size, biocompatibility and straightforward manner in which they could be programmed to interact with cellular nucleic acids through hybridization. However, to realize such applica-tions using tools from DNA nanotechnology, it will be necessary to bridge the gap between performing experiments in well-mixed reaction buffers and spatially structured, densely packed cellular environments (Box1).

In this Review, we summarize recent progress towards the goal of bringing DNA nanotechnology into the cell. We focus on nucleic acid nanodevices and nanostructures that are rationally designed, chemically synthesized and then delivered to mammalian cells. We begin with a brief overview of DNA nanotechnology in cell-free settings, and then move to more cell-like environments, such as cell lysates and fixed cells settings that capture some, but not all, of the complexity of cellular environments. Next, we discuss

DNA nanotechnology from the test tube to the cellYuan-Jyue Chen1, Benjamin Groves1, Richard A. Muscat1 and Georg Seelig1,2*

The programmability of WatsonCrick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, research-ers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mecha-nisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology applications that best take advantage of the small size, biocompatibility and program-mability of DNA-based systems lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

several recent results that show how DNA nanodevices can be pro-grammed to interact with cell surface proteins, before turning to work on the delivery of DNA devices and structures into cells. We reach devices that operate inside live cells and review initial work towards using DNA sensors and logic gates to detect, analyse and regulate cellular RNA levels. We put this work into context by high-lighting design principles identified in the development of live-cell RNA imaging probes, small interfering RNAs (siRNAs) or anti-sense oligonucleotides (ASOs), which could be used to improve the performance of DNA devices in cells. Finally, we make connections to RNA nanotechnology and RNA synthetic biology, which have broadly similar aims to DNA nanotechnology but typically rely on the use of genetically encoded and transcribed RNA.

Cell-free DNA nanotechnologyTo operate reliably in complex, wet environments, living organ-isms use molecular sensors to detect changes in that environment, motors and actuators to adapt to the environment, computational control circuits to convert sensor information into motor activity, and structural elements that protect and organize these components. Intriguingly, cell-free DNA nanotechnology has made progress towards the construction of most of the functional components both structures and dynamic devices required for creating molec-ular robots that can emulate some of the behavioural complexity observed in biology. Here we review a few key results from cell-free DNA nanotechnology and point out potential applications in the cellular environment.

Structural DNA nanotechnology. In the 1980s, Nadrian Seeman developed the notion that DNA could be used as a structural engineering material810. In 1998, Winfreeetal. provided the first experimental demonstration of large-scale structure formation: they showed that micrometre-sized periodic DNA lattices could self-assemble from nanoscale DNA tiles that are themselves assem-blies of multiple oligonucleotides11. Subsequently, tile assembly and related techniques were successfully used to create a wide variety of lattices and wireframe DNA structures1119.

Rothemund further advanced structural DNA self-assembly by developing DNA origami, a technique that is easy to use, flex-ible enough to accommodate almost any two-dimensional (2D)

1Department of Electrical Engineering, University of Washington, Seattle, Washington 98195, USA. 2Department of Computer Science and Engineering, University of Washington, Seattle, Washington 98195, USA. *e-mail: [email protected]

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structure of interest, and reliably results in a high yield of the target structure20. DNA origami relies on the use of a long single-stranded scaffold strand that is folded into a target structure through hybridi-zation with a large number of short staple strands. This technology was rapidly and broadly adopted, and was soon generalized to the self-assembly of three-dimensional (3D) structures2124. DNA nano-structures are beginning to be investigated as tools for drug delivery and similar applications because they provide precisely programm-able scaffolds for the attachment of functional groups including drug and targeting moieties, and because 3D structures can be designed to act as protective enclosures for a cargo of interest.

Dynamic DNA nanotechnology. Dynamic DNA nanotechnology combines self-assembly through programmed hybridization with DNAzyme catalysis or DNA strand displacement reactions a form of competitive hybridization to create devices with moving parts and time-varying behaviours. Dynamic DNA nano technology can be traced to multiple sources, including Adlemans work on DNA computation and research on the directed evolution and characteri-zation of functional nucleic acids25. However, Yurke and co-workers truly launched the field by demonstrating that a functional molecu-lar motor could be rationally designed and driven through its work cycle using only hybridization and strand displacement reactions26. Subsequently, the Winfree and Pierce groups demonstrated that multiple strand displacement reactions could be chained together to create complex reaction cascades27,28. Owing to their simplicity, DNA strand displacement cascades have since been used widely and effectively for molecular engineering and provide the mechanism that drives most dynamic DNA devices.

Dynamic DNA nanotechnology has resulted in molecular motors29,30, including walking motors that autonomously move along a track3134, molecular circuits that can analyse information encoded in complex mixtures of molecules27,3539, and catalytic amplifiers that can sense and amplify signals4044. Many of these systems have obvi-ous potential for biotechnological applications: for example, Shapiro and collaborators used DNA and a restriction enzyme to build a molecular automaton that could diagnose the state of a disease by detecting and analysing a set of molecular markers, thus realizing, in a test tube, a type of computation similar to those performed by gene regulatory networks6,45. Conversely, the analysis and manipulation of molecular information in and on living cells is the one area of appli-cation in which molecular devices and structures can out perform their electromechanical counterparts.

DNA nanotechnology in lysates and fixed cellsCellular conditions are significantly different from those used in cell-free experiments (Box1): the presence of nucleic-acid-binding proteins, including DNases and RNases, may interfere with device performance. Moreover, cellular environments are highly structured, which inhibits the free diffusion of exogenously delivered nucleic acids. Cell lysates, serum and fixed cells provide reaction environ-ments that each capture some of the complexity of live cells and ena-ble testing and optimization of nucleic acid devices in comparably well-controlled conditions.

Stability of DNA nanostructures in cell lysates and serum. Lysates are mixtures of cellular components created from cells that have been homogenized. Because lysates lack any kind of cell wall, nucleic acid devices can readily be placed into an environ-ment imitating that found inside the cell, although the concentra-tions and activities of the cellular components encountered by the DNA nano structure are usually different. Yan, Meldrum and col-laborators tested the stability of DNA origami in cell lysate and found that origamis could be extracted from the lysate and char-acterized following up to 12hours of incubation46. In contrast, long single- and double-stranded nucleic acids could not be recovered

after incubation. Because detailed conditions for mixing the ori-gami with cell lysate were not reported, it is difficult to evaluate how closely the reaction buffer approximated physiological conditions. Furthermore, because DNA nanostructures are typically assembled in buffers with high Mg2+ concentrations (~10mM), the addition of large amounts of nanostructures could increase the Mg2+ level, thus making the structures seem more structurally robust than what might be expected in a cell. Still, such effects can be controlled, and lysates constitute a useful setting for exploring how nanodevices might fare in biologicalenvironments.

Moving nanostructures into cell culture and animals will require devices that are stable in the presence of serum and serum- supplemented media. Like lysates, serum contains nucleases and lacks stabilizing salts such as magnesium. Conway et al. showed that small three-stranded nanostructures in the shape of a triangu-lar prism were more stable in serum than the individual compo-nent strands47. A gel analysis showed that individual strands had a half-life of less than an hour in 10% fetal bovine serum, whereas the half-life of intact structures was closer to two hours. The use of chemically modified strands resulted in structures with half-lives even longer than 24 hours.

In a comprehensive analysis, Perrault and colleagues tested three different 3D origamis in mammalian cell culture media sup-plemented with serum, and showed that the structural integrity of origamis is strongly dependent on the origami design, the presence

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Figure 1 | Applications of DNA nanotechnology at the interface with biology. a, Smart therapeutics could combine structural elements with molecular logic to target therapeutic actions to a specific cell or tissue type, thus minimizing side effects60. b, DNA nanostructures can serve as programmable scaffolds for attaching drugs, targeting ligands and other modifications, such as lipid bilayers78. c, A novel class of sensitive and specific imaging probes that takes advantage of DNA-based amplification mechanisms can be programmed to sequence-specifically interact with cellular RNA52. d, DNA origami and other structures provide precise control over the spatial organization of functional molecular groups, which makes them intriguing tools for quantitative measurements in cell biology66. Figure reproduced with permission from: a, ref.60, AAAS; b, ref.78, American Chemical Society; c, ref.52, American Chemical Society; d, ref.66, Nature Publishing Group.

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of Mg2+ and the level of nuclease activity48. Using gel and transmis-sion electron microscopy assays, they observed that two of their three test constructs were partially denatured after incubation in cell culture media for a day, and that addition of 6 mM Mg2+ to the media was required to inhibit this effect. Intriguingly, a third structure, an origami nanotube, was structurally stable even with-out the added salt. After 24 hours, all three structures were partially degraded by DNases when >5% fetal bovine serum was added to the media. Importantly, nuclease degradation could be dramatically reduced by addition of actin, a protein that binds competitively to nucleases; this modification was found to be compatible with cell culture conditions.

To quantify the degree of nanostructure degradation by DNases, Keum and Bermudez measured the half-life of wireframe tetra-hedral DNA nanostructures (TDNs) in the presence of DNase 1. They found that TDNs were up to three times more stable than double- stranded DNA49. Likewise, DNA origami has also been shown to be more stable than duplex DNA in the presence of nucle-ases. Castroetal. incubated DNA origami with different nucleases and assessed origami degradation after 24 hours using transmission electron microscopy and gel assays50. Degradation was observed in the presence of DNase 1 and T7 endonuclease 1; however, at least for DNase 1, the measured rate of degradation was several hundred-fold slower than for duplex DNA. Comparing this result with the TDN study suggests that DNA origami are even more sta-ble than smaller wireframe TDNs, perhaps because they are more highlyinterconnected.

Together, these results suggest that DNA nanostructures can withstand degradation by nucleases considerably better than sim-ple single- and double-stranded nucleic acids. Moreover, some

nanostructures can retain their structural integrity over extended time periods in physiological salt conditions. Further research is required to understand fully the interplay between the functionality of a given structure and its stability.

DNA nanotechnology in fixed cells. Permeabilized cells and tis-sues also mimic some aspects of the cellular environment: fixed cells retain much of their structural organization, in particular the spatial distribution of mRNA and proteins. Fixed cells provide a controlled setting for visualizing the subcellular distribution of mRNAs and proteins using immunostaining or fluorescence in situ hybridiza-tion (FISH); approaches from DNA nanotechnology have already proved practically useful for increasing the sensitivity and specific-ity of such imaging methods. For instance, molecular probes based on a hybridization chain reaction (HCR)28 have enabled the simulta-neous mapping of up to five target mRNAs within intact vertebrate embryos51,52. By hybridizing a set of adaptor strands to target mRNA sequences, Choietal. were able to controllably catalyse a polym-erization reaction of two types of fluorescently labelled hairpin monomers; as a result of this catalytic hybridization reaction, the fluorescent signal associated with a given mRNA is amplified and can be imaged readily using a fluorescence microscope (Fig.2a).

By combining the ideas of strand displacement with single-molecule FISH (smFISH), Raj and colleagues were able to detect single-nucleotide variations within individual mRNA transcripts53. When performing smFISH, a collection of singly labelled DNA oligo nucleotides hybridize along the target RNA transcripts in fixed cells54. Co-localization of multiple probes on the same transcript produces a discrete fluorescence spot that is clearly discernable using conventional fluorescence microscopy. Discrimination at the

The different techniques, design considerations and limitations discovered by researchers working with ASOs, siRNAs, molecu-lar beacons and related technologies help us highlight some of the challenges of bringing nucleic acid nanotechnology to the cellularenvironment.

Delivery. In test tubes, the concentration of all components can be precisely controlled and reaction kinetics can be monitored with high time resolution. In contrast, to function in cells, nucleic acid devices must first cross the cell membrane. Different nucleic acid delivery methods can result in vast differences in cellular uptake timing, amount and subcellular distribution, and even cell viabil-ity. For example, commonly used lipid-based transfection reagents efficiently deliver large numbers of probes to cells, but a signifi-cant fraction are enclosed in endosomes and thus do not reach the cytoplasm132,133. Conversely, microinjection can deliver nucleic acids directly to the cytoplasm or nucleus, but is limited to a rela-tively small number of cells. We refer the reader to Baoetal.134 for a more in-depth comparison of different methods for the delivery of synthetic nucleic acids to cultured cells, and to Davisetal.85 for a review of nanoparticle-based drug delivery.

Stability and chemical modifications. The cellular half-lives of short, unmodified nucleic acids are of the order of minutes135. However, a number of chemical modifications to the sugar, base and backbone of nucleic acids have been identified that dramatically enhance their stability. The most commonly used modifications include phosphorothioate inter-nucleotide linkages and 2O-methyl ribose modifications136. Because chemical modifications provide efficient ways to protect nucleic acids against degradation by nucle-ases, some of them (for example, phosphorothioate bonds137) also

tend to have adverse effects on cell viability138. Therefore, when choosing modifications for nucleic acid devices, it is important to strike a balance between device stability and cell viability.

Molecular crowding and cellular compartmentalization. Cells are densely packed with proteins and other macromolecules, which can adversely affect the performance of multi-stage, multi-input logic circuits and other systems with many interacting com-ponents. The diffusion coefficient of synthetic DNA molecules in the cytoplasm is 5100 times smaller than in water, depending on the size of the molecule139. The rates of hybridization between com-plementary single-stranded nucleic acids are also different in the cellular environment than in an aqueous buffer140. Furthermore, mammalian cells are organized in a variety of different compart-ments, and enclosures within such compartments could prevent distinct circuit components from encountering each other.

Immune activation. Exogenously delivered nucleic acids can trig-ger an innate immune response through the activation of Toll-like receptors (TLR). TLR3 responds to double-stranded RNA; TLR7 and TLR8 respond to single-stranded RNA; and TLR9 responds to unmethylated cytosine-guanine (CpG) motifs in DNA. TLR9 serves to detect DNA of bacterial origin by exploiting the fact that in mammalian cell genomic DNA the dinucleotide CpG is generally methylated, whereas in bacteria it is not79. Double-stranded RNA longer than 30 bp is bound by protein kinase R, which activates a cellular immune response that can result in cell death141. Activation of TLR9142- or PKR143-mediated responses are considered for therapeutic applications where immune stimula-tion may be desirable. However, it is more common to avoid such immunestimulation.

Box 1 | Synthetic nucleic acids in the cellular environment.

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level of individual nucleotides was achieved using an additional strand displacement probe modified with a distinct fluorophore/quencher pair53. Probe binding through toehold-mediated strand displacement was dramatically slowed in the presence of a mis-match between the toehold and target. Co-localization of the single-nucleotide variation probe with the transcript signal was used to verify the identity of the sequence (Fig.2b).

Strand displacement in fixed cells has also been demonstrated for DNA-tagged proteins55. The ability to hybridize and displace strands

means that the number of protein species that may be imaged is no longer constrained by the number of resolvable wavelengths avail-able to the microscope (generally around four), but is instead lim-ited only by the number of sequential hybridization/displacement cycles that may be performed.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is an approach that similarly takes advantage of the reversibility of DNA hybridization. Short, fluorescently labelled DNA imager strands are used to bind transiently to complementary

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Figure 2 | In situ imaging of mRNA in fixed cells. a, HCR FISH52. Left: Initiator strands I1 and I2 hybridize to a target mRNA, which triggers a polymerization reaction between the two fluorescently labelled hairpin monomers H1 and H2. As a result, the target mRNA is connected to multiple fluorophores and can be visualized using fluorescence microscopy. Right: Confocal microscopy images at different z planes in a fixed zebrafish embryo. HCR probes are used to identify four different mRNAs (red: Tg(flk1:egfp); blue: tpm3; green: elevl3; yellow: ntla). b, Detection of a single-nucleotide variation using strand displacement probes53. Left: Reaction mechanism. Mutant and wild-type probes compete for binding to a target mRNA. Because binding kinetics strongly depend on toehold sequence, each probe type primarily binds to the cognate mRNA. Co-localization of single-nucleotide variation detection probes with multiple mRNA-targeting guide probes further shows that the signal is indeed triggered off the mRNA. Right: Fluorescence micrographs of BRAF mRNA detected using guide probes (image 1), wild-type probes (image 2) and mutant probes (image 3). Image 4 shows mRNA classified as wild type or mutant. SNV, single-nucleotide variation. Figure reproduced with permission from: a, ref.52, American Chemical Society; b, ref.53, Nature Publishing Group.




docking strands attached to a target56. The spontaneous binding and unbinding causes the fluorescence at a given point to switch between the on and off state, thus allowing individual target sites to be imaged with sub-10-nm resolution using total internal reflection microscopy. As above, the reversible nature of DNA-PAINT means that it is not limited by the number of fluorophores, and sequen-tial labelling allows the reuse of fluorescent dyes. DNA-PAINT was adapted to the insitu 3D imaging of fixed cells by targeting cellular proteins with antibodies conjugated to DNA docking strands57.

Interacting with cell surface markersMammalian cells are comprised of a number of compartments and structures, which all act as discrete vessels for biochemical reac-tions. The most accessible structure is the cell surface itself a lipid bilayer incorporating many surface proteins that often differenti-ate one cell type from another. Several recent papers demonstrated that DNA nanosystems can be designed to interact with cell surface markers; as with antibodies and aptamers58, the most mature exam-ples of potential DNA-based therapeutics target cell surface mark-ers and cells in the bloodstream targets that do not require the uptake of nanostructures into specific cells and tissues.

Stojanovic and colleagues directed the probes to particular cell surface proteins by covalently attaching DNA strand displacement

probes to protein antibodies59. Cells were labelled with one or two probes, depending on which proteins were displayed on the cell surface. After the binding stage, a trigger strand was added to acti-vate a strand displacement cascade involving the attached probes. The output of the cascade depended on whether one or both probes were present, thus allowing the cell types to be distinguished (Fig.3a). Although a similar outcome could be achieved by directly labelling cells with two fluorescently tagged antibodies, this work demonstrates a more easily scalable approach for performing cell-state classification, potentially allowing many molecular markers to be analysed in parallel and information to be summarized into an easy-to-interpret actionable signal.

Douglasetal. created a DNA nanorobot capable of delivering a molecular payload to particular cell types60. The payload was enclosed by a hinged origami container, which was initially held in a closed conformation to sequester the cargo. Aptamers DNA or RNA sequences selected to bind specific proteins or even whole cells61,62 provided the means of targeting the nanorobot to specific cells with-out the need for covalent attachment of DNA strands to antibodies. The same aptamers were also part of the locking mechanism; aptamer binding to the target protein triggered a conformational change, thus allowing the origami lid to open and expose the cargo. AND logic implemented by employing combinations of two different

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Figure 3 | Cell surface computation. a, In situ cell classification by evaluating specific surface markers59. Cells are first coated with DNA-modified antibodies (DNA circuits; antibodies are shown as rectangles or ellipses, DNA strands as coloured lines), and depending on the surface marker profiles of the cell type, either one or two gates can bind to cells. The subsequent introduction of an initiator strand (red) triggers a series of strand displacement reactions (fully complementary strands share the same colours). A soluble reporter complex can fluorescently tag only cells labelled with two surface-bound gates. b, Molecular robot for targeting a therapeutic action to specific cell types. The schematic shows how a barrel-shaped nanorobot responds to specific antigens (keys) expressed on cells surfaces60. The nanorobot is initially held in a closed configuration by two aptamer locks; only when it encounters a cell that displays two matching antigens can it be opened, thereby exposing a drug. Bottom: Transmission electron microscopy images of the closed and open states of the nanorobots (scale bars, 20nm). Figure reproduced with permission from: a, ref.59, Nature Publishing Group; b, ref.60, AAAS.




aptamers was used to target distinct fluorophores or a drug mol-ecule to a subset of cells (Fig.3b). Intriguingly, a similar nanorobot was also shown to be active in the bloodstream of live cockroaches, with multiple robot species performing logic operations63.

The Tan group has also used aptamer-based logic gates to dis-tinguish cells in mixed populations64. In this implementation, a scaffold is used to link a logic gate with multiple aptamers. Binding the aptamers to surface proteins releases DNA strands that act as inputs to the logic gate. Thus, the logic gates will only be triggered if the appropriate aptamer ligands are present on the surface of the cell. Crosstalk between cells is minimal, which suggests that nearby interactions are preferential owing to a higher local concentration of interacting species. The same group has recently demonstrated a more modular gate design that allows for a greater number of inputs and the potential for combining with the scaffold approach to improve off-target effects65.

Not only can DNA devices interrogate combinations of markers present on a cell surface, but the nanometre-precision addressability of an origami can be used to control the density of ligands for cell surface receptors66. Shawetal. constructed DNA nanocalipers by arranging ephrin-A5 ligands on a DNA origami scaffold. By taking advantage of the positional addressability possible with DNA ori-gami, they showed that cells are sensitive to the spatial arrangement of ligands.

The addressability of DNA systems can also be used to imbue cells with a novel identity. Francis and colleagues developed a method for affixing synthetic single-stranded DNA strands to liv-ing cells, thereby allowing an unlimited number of coding specifici-ties67. Cells were treated with a synthetic sugar that was metabolized and integrated into the cell membrane where they could then serve as chemical handles. Phosphine-modified single-stranded DNA molecules were then attached to the handles through Staudingerligation68.

Gartner and Bertozzi showed that this approach can be used to organize cells into 3D multicellular assemblies69. As a demonstra-tion of structure guiding the function of a tissue, the authors gen-erated a microtissue using assemblies of Chinese hamster ovary cells expressing external growth factor interleukin-3 (IL-3) to sup-port the growth of haematopoietic progenitor cells (FL5.12 cells); growth of the progenitor cells only occurred when they were associ-ated with the IL-3 secreting cells. The Gartner group went on to use this strategy to investigate the effect of cell-to-cell variation in Ras signalling on the morphogenesis of microtissues70.

In addition to mimicking cellcell adhesion moieties, DNA can also be used to imitate other protein functions. Through modifica-tion with hydrophobic groups, DNA structures can be inserted into lipid membranes71,72. Burnsetal. demonstrated that a DNA nano-pore channel inserted into the membrane of mammalian cells has a number of cytotoxic effects73, possibly due to the free movement of critical ions, nutrients and other molecules across the membrane.

DNA nanostructures as drug-delivery vehiclesThe work discussed so far not only demonstrates the feasibility of operating DNA nanodevices and structures in cell lysates and cell culture (and in insects), but also shows that nanosystems can inter-act with cell surface proteins. We now move on to review the chal-lenges associated with the delivery of nanodevices into mammalian cells, and also discuss their use as vehicles for drug delivery.

Cellular uptake of DNA nanostructures. By engineering folate-conjugated DNA nanotubes to target the folate receptors that are overexpressed on many cancer cells, Mao et al. successfully dem-onstrated that large DNA nanostructures can enter cells. They also further modified the nanotubes with a fluorescent label to confirm that the nanotubes, or at least nanotube fragments, were internalized upon receptor binding7.

Walshetal. demonstrated that the uptake of TDNs into human embryonic kidney cells was similarly efficient with or without a lipid-based transfection reagent. A Frster resonance energy trans-fer (FRET) assay was used to demonstrate that TDNs remain intact for a long time after cellular uptake74. Work by Schulleretal. showed that DNA origami structures much larger than TDNs could also enter cells without the need for transfection reagents75. Internalization of a fluorescently labelled strand attached to the origami made it pos-sible to visualize origami uptake, although this assay could not be used to determine whether the origami structures were still intact in the cell.

Given the anionic nature of DNA, it is surprising that cells take up DNA nanostructures in the absence of transfection reagents. Liang et al. recently investigated the mechanism responsible for TDN uptake and found that they enter mammalian cells through receptor-mediated endocytosis, specifically the caveolin-dependent pathway. Once inside cells, TDNs are actively transported along microtubules and eventually accumulate in lysosomes. To dem-onstrate that DNA nanostructures are capable of targeting differ-ent intracellular locations, Liang et al. coupled TDNs to nuclear localization signal peptides, thus successfully directing them to thenucleus76.

Even though DNA nanostructures can enter cells with surprising efficiency, additional modifications can be used to further enhance uptake or increase stability. For example, Mikkilaetal. demonstrated that rectangular DNA origami coated with viral capsid proteins were taken up by human embryonic kidney cells at an efficiency ten times that of the same origami delivered with Lipofectamine 200077.

Perrault and Shih constructed DNA nano-octahedrons encap-sulated in a lipid bilayer. By incorporating lipid-coupled DNA oligos, the octahedron served as a template for the formation of a surrounding lipid shell78. The encapsulated octahedrons showed reduced immune activation and dramatically enhanced bioavail-ability in circulation in mice, compared with non-encapsulated controls (Fig.1b).

Drug delivery. CpG oligodeoxynucleotides (ODNs) are DNA sequences with unmethylated cytosine-phosphate-guanine stretches that can trigger a strong innate immune response by activating the Toll-like receptor TLR979. CpG ODNs are an attractive thera-peutic cargo because they can be integrated directly into any DNA nanostructure through hybridization. Takakura and co- workers engineered Y-shaped DNA with CpG motifs to trigger immune responses80,81. They found that Y-shaped DNA, compared with con-ventional single- or double-stranded DNA, are more efficiently taken up by macrophage cells, thus enhancing immune stimulation. Later works have demonstrated efficient uptake and the activation of an immune response with multi-arm82, TDN83 and origami structures75 functionalized with multiple CpG ODNs.

Chang and co-workers created a synthetic vaccine complex by assembling TDNs that were modified with both streptavidin and CpG ODNs84. Streptavidin served as a model antigen, whereas the CpG ODN was an adjuvant used to enhance the immune response. The construct was first tested in a mouse macrophage-like cell line and then injected into mice. Mice injected with the fully assembled complex developed higher levels of anti-streptavidin IgG anti bodies than control mice injected with a simple mixture of streptavidin andCpG.

DNA nanostructures have also been designed to serve as car-riers for doxorubicin, a cytotoxic drug that is used in a variety of cancer therapies. Previous work has shown that using nano-particles to package doxorubicin could reduce side effects and dramatically increase circulation time in the body85. Moreover, because doxorubicin intercalates in DNA, it is a natural match for delivery with a DNA vehicle an idea first introduced in the DNA aptamer field58.




Changetal. were the first to use DNA nanostructures specifi-cally wireframe icosahedra to deliver doxorubicin to cancer cells86. Jiangetal. built on this idea and created DNA origami triangles and tubes to carry doxorubicin into human breast adenocarcinoma cancer cells. The much larger size of the origami structures further enhanced the amount of doxorubicin that could be delivered, and the drug-loaded complexes showed toxicity not only in regular can-cer cells, but also in doxorubicin-resistant cancer cells87. Kimetal. similarly found that the TDN-based delivery of doxorubicin resulted in drug activity in an otherwise drug-resistant cell line88. When employing DNA origami nanotubes with varying degrees of global twist, Hogberg and collaborators found that doxorubicin loading, delivery and release could be tuned by changing the amount of twist in the structure89. Zhuetal. constructed a DNA polymer based on an HCR-like mechanism to deliver doxorubicin90. The polymers could specifically target cancer cells via the recognition ability of an aptamer, inhibiting tumour growth in mice. Finally, after injecting DNA origami triangles loaded with doxorubicin into the tail veins of tumour-bearing mice, Zhangetal. found that origami-based deliv-ery resulted in a faster reduction of tumour mass than delivery of equal amounts of doxorubicin without a DNA origami carrier91.

Short interfering DNA (siRNA) and ASO delivery have also been explored. By inserting a DNA loop containing an antisense sequence into one edge of a TDN, Keumetal. showed that the ASO reduced protein expression through RNaseH-mediated degradation of a target mRNA in a sequence-specific manner92. TDN-mediated siRNA delivery to tumours in a mouse model was demonstrated by Leeetal.93, who hybridized siRNA to TDNs that were also conju-gated to cancer-targeting ligands. They showed that in cell culture, both the number and relative orientation of ligands affected uptake and gene-silencing efficiency. The ligand-coupled TDNs exhibited high tissue specificity in mice, accumulating mostly in the kidney and the tumour, but negligibly in other organs; it should be noted that the structures used invivo differed in the number of attached ligands from those characterized invitro. Moreover, it remains diffi-cult to judge how robust the structures were in such an environment as no data was provided on the stability of the structures invivo. Finally, Weizmann and collaborators created a periodic origami nanoribbon using a scaffold strand obtained by rolling circle ampli-fication. Nanoribbons entered cells through a clathrin-mediated pathway, and siRNA covalently attached to the structure resulted in better knockdown than siRNA that were simply mixed in with thenanoribbons94.

The work discussed in this section outlines an important proof-of-principle: DNA nanostructures can serve as drug-delivery vehicles. What sets DNA structures apart from approaches based on nano-particles or polymers is primarily the programmability of a DNA scaffold, but also the very high degree of shape and size uniformity that can be achieved.

Dynamic DNA nanodevices inside living cellsIn this section, we review work on dynamic nucleic acid devices that operate within cells and respond to specific environmental cues; this includes devices that sense global environmental variables such as pH, and recent progress towards detecting specific molecu-lar information carriers such as cellular RNA. Finally, we discuss how the output of a nucleic acid device could allow modulation of gene expression levels and review first steps towards the con-struction of logic circuits for the detection and analysis of multiple molecularmarkers.

Sensing the cellular environment. Functional nucleic acids such as DNAzymes and aptamers have been used extensively within the biosensor field to detect the levels of various molecular species within cells. Here we will highlight two results that combine such sensing moieties with DNA nanomotors or structures.

Peietal. constructed a set of TDNs with one or two reconfigur-able edges, which allowed the TDN to change its shape in response to specific molecular signals such as protons, ATP and mercury ions95. Using a FRET reporter strategy, they showed that a recon-figurable TDN changed conformation in response to intracellular ATP. This demonstrated the feasibility of combining a DNA nano-structure with cellular sensors, which is an important property for any potential smart drug.

Along a similar line, Modi et al. used a DNA-based sensor to map the pH of endosomal pathways in living cells96. The design was based on Yurkes DNA tweezers essentially two double helices connected with a flexible hinge but incorporated an i- quadruplex structure that acts as a pH-sensitive switch to open and close the tweezers (Fig. 4a). The sensor was taken up by fly haemocytes through endocytosis and trafficked from early endosomes (pH~6) to late endosomes (pH ~5.5), and finally to lysosomes (pH ~5). The increasingly acidic environment resulted in quantifiable fluo-rescence changes and thus an indirect measurement of the pH. Coupling the sensor to the protein transferrin allowed the pH changes to be mapped along a specific receptor-mediated endo-cytic pathway. In a follow-up study, the same group showed that

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Figure 4 | DNA nanomachines and logic gates in mammalian cells. a,b, pH-sensitive DNA nanomachines for simultaneously probing the furin (Fu) and transferrin (Tf) pathways97. Left: A transferrin-modified DNA nanomachine TfITf is confined to the transferrin pathway. The nanomachine enters a sorting endosome (SE), then a recycling endosome (RE), and eventually returns to the membrane. The DNA nanomachine FuIFu targets the furin pathway: it enters the SE, then late endosome (LE), and eventually localizes in the trans-Golgi network (TGN). Nanomachine fluorescence is sensitive to pH, which varies between different endosomal compartments. Right: pH-sensitive elements of DNA nanomachines IFu (green strands, top) and DNA nanomachines ITf (pink strands, bottom) form i-motif at low pH, which causes high FRET between the two fluorophores. b, A DNAzyme-based AND logic gate operates inside living cells113. Left: Synthesized inputs with the sequences of miR-21 and mir-125b are micro-injected together with the logic AND gate. Right: Reaction mechanism. Input B first binds to the hairpin (green segment), which is then available to interact with input A to join the two components of the AND gate. The joined DNAzyme complex can then cleave the substrate, thus leading to high fluorescence by separating a fluorophore (red dot) from a quencher (black dot). Figure reproduced with permission from: a, ref.97, Nature Publishing Group; b, ref.113, Nature Publishing Group.




pH-sensitive nanomachines could simultaneously track multiple pathways in the same cell97.

Sensing cellular RNA. The identity and health of a cell can often be inferred by its RNA repertoire. However, the detection of specific cellular nucleic acids can be challenging because it requires nanode-vices to access the cytoplasm, where most mature mRNA or miRNA are located. Furthermore, the low copy number of many RNA spe-cies may make signal amplification necessary, and the secondary structure or RNA-binding proteins can reduce the accessibility of certain sequences.

The live cell imaging field has addressed many of these issues and has developed a number of nucleic acid probes for detecting specific mRNA sequences in live cells. Chemical and structural modifications used to improve the performance of live-cell imaging probes could also be used to enhance the intracellular performance of DNA nanodevices. Molecular beacons stem-loop probes with a fluorophore and quencher attached to the stem are probably the best-studied class of probes for detecting mRNA in living cells98. Fluorescence is quenched when the probe is delivered but becomes unquenched when the probe hybridizes to the target mRNA. Variations on this basic design have resulted in improved perfor-mance: the addition of a longer double-stranded RNA domain or

a transfer RNA (tRNA) sequence resulted in active export of the probe from the nucleus to the cytoplasm, thus facilitating interac-tions with mRNA99101 (Fig. 5a). Chemical modifications, in par-ticular 2OMe RNA, have been used to enhance probe stability by protecting against degradation by cellular nucleases102. As in the single-cell FISH techniques described above, co-localization of multiple probes on the same transcript can improve the signal-to-background ratio101. Multivalent probe designs such as MTRIPs, in which several linear oligonucleotides are attached to a streptavidin core, can also result in a stronger signal103 (Fig.5b).

Nanoflares, developed by Mirkin and co-workers, provided the first example of a DNA strand displacement reaction with an RNA input in live cells (Fig.5c). Nanoflares consist of gold nanoparticles function-alized with DNA oligonucleotides complementary to an mRNA or miRNA target. Shorter fluorescently labelled oligos are hybridized to the nanoflares and quenched by proximity to the gold nanoparticle104. Binding to the target displaces the fluorescently labelled strand, which results in increased fluorescence. A modified version of the nanoflare technology used an LNA-modified oligonucleotide to increase bind-ing strength with RNA targets while simultaneously targeting them for degradation105,106. Haloetal. further demonstrated that nanoflares, in combination with flow cytometry, can be used to distinguish live circulating tumour cells in the context of whole blood107.

ba

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Figure 5 | mRNA imaging in living cells. a, Ratiometric bimolecular beacons (RBMBs)101. Top: Binding to a target mRNA separates the reporter dye (red dot) from the quencher (black dot), which results in high fluorescence. Multiple RBMBs can bind to the tandem repeat targets in the 3UTR of a heterologous mRNA, thereby enabling visualization of a single transcript in living cells. A reference dye (pink dot) is used to control cell-to-cell variation in molecular beacon delivery. Bottom: Fluorescence images of HT1080 cells using RBMB and FISH probes for the same mRNA. Image 1: Fish probes; Image 2: RBMB reporter dye; Image 3: A merged image that also includes nuclear DAPI stain (blue). b, Multiply labelled tetravalent RNA imaging probes (MTRIPs)103. Top: MTRIPs consist of multiple fluorophore labelled oligonucleotides attached to streptavidin (purple). Multiple MTRIPs can be designed to hybridize to a target mRNA, thus making single mRNA visible in living cells. Bottom: Deconvoluted confocal microscopy images of individual -actin mRNA in an A549 cell. Image 1: MTRIPs; Image 2: Scrambled probes; Image 3: A merged image that includes nuclear DAPI stain. c, Nanoflares104107. Left: A nanoflare contains long capture strands and fluorophore-labelled flare strands, which are initially quenched by the gold nanoparticle. Target mRNAs can bind to capture strands, displace the flare strand and trigger an increase in fluorescence. Right: Confocal fluorescence microscopy images of HeLa cells treated with either control nanoflares (left) or Survivin (target mRNA) nanoflares (right). Figure reproduced with permission from: a, ref.101, Oxford Univ. Press; b, ref.103, Nature Publishing Group; c, ref.106, American Chemical Society.




Modulating cellular RNA. Existing non-coding nucleic acid tech-nologies for gene-regulation, such as ASOs, ribozymes and siRNAs, not only inform the design of cellular DNA nanosystems, but could also be integrated with DNA nanodevices as a means of controlling the cellular environment.

Afoninetal. demonstrated the assembly of a functional siRNA from two DNA:RNA complexes that individually did not enter the RNAi pathway in cells108. The reaction was initiated by hybridiza-tion of complementary single-stranded RNA overhangs present in the two inactive complexes and is likely to have proceeded through four-way strand exchange109. siRNA activity was observed in cell culture and tumour xenograft mouse models.

It would be even more intriguing if the activation of a regula-tory response could be conditional on the detection of a specific molecular marker. Benenson and colleagues took a first step in this direction by designing nucleic acid displacement circuits that interact with components of a cell lysate110: detection of an RNA sequence added to the lysate triggered a strand displacement reac-tion, leading to the creation of a functioning siRNA. Pierce and collaborators demonstrated a more general mechanism for the conditional formation of a Dicer substrate RNA in a cell-free bio-chemical assay111. Yokobayashi and co-workers built a genetically encoded RNA hairpin system that formed a substrate for the RNAi pathway upon activation by a synthetic, exogenously delivered inputoligonucleotide112.

Molecular computation. The realization of multi-input, multi-layer molecular circuits is one of the major accomplishments of dynamic DNA nanotechnology. But what unique advantages would DNA nanotechnology bring to engineering cellular biocomputers, com-pared with alternative technologies based on synthetic gene regu-latory networks? First, DNA circuits rely on components that are mechanistically simple and rationally designed at the molecular level, which provides a high degree of control over the reaction path-way. Second, new, orthogonal components can be designed simply by changing sequence, which makes it easy to increase system size in a modular fashion. Third, most dynamic DNA devices have a relatively small DNA footprint compared with systems assembled from genetically encoded proteins.

The Shapiro group microinjected a DNAzyme AND gate along with miRNA-derived inputs into MCF7 breast carcinoma cells113 (Fig.4b). The gate was protected from nucleases by the addition of inverted thymidine groups to the 3 ends. Gate activation was quan-tified using fluorescence microscopy and, consistent with AND logic, fluorescence increased only in the presence of both inputs.

Strand-displacement DNA logic gates have also recently been used to detect combinations of miRNA in living cells. Based on designs first demonstrated in vitro27, Hemphill and Deiters used an AND gate constructed from DNA to detect the endogenous miRNAs miR-122 and miR-21 in Huh7 hepatocellular carcinoma cells114. Gates were delivered using standard transfection reagents and gate activation was observed only in cells that produced both input miRNA.

The results reviewed in this section suggest that the field is mak-ing rapid progress towards the design of dynamic DNA devices that can sense information in cells, analyse that information using embedded molecular control circuits and then respond by effecting changes in the cell.

Genetically encoded structures and devicesApplications from gene therapy to metabolic engineering require long-term embedded control of gene expression. RNA scaffolds and regulatory elements that can be genetically encoded and transcribed in living cells are likely to be a better match for such applications than transiently delivered synthetic DNA systems. Modifying existing DNA nanotechnology to be compatible with

transcription requires extensive adjustments to the experimental approach and molecular design. Although this may seem like a sig-nificant challenge, considerable progress has already been made. Here we highlight a few intriguing results using transcribed RNA systems that straddle the increasingly blurred line between nucleic acid nanotechnology and synthetic biology.

Cell-free RNA nanotechnology has resulted in a variety of 2D and 3D structures, and the size of structures that can be realized with RNA is rapidly increasing115120. For example, Afonin et al. demonstrated co-transcriptional, isothermal assembly of RNA cubes made from six or ten ~40bp strands120. Even more recently, Geary, Rothemund and Andersen demonstrated the feasibility of the computational design and experimental implementation of co-transcriptional folding of RNA origami tiles up to 660 nucleotides in size121. These tiles also self-assembled into larger lattice structures, with dimensions reaching hundreds of nanometres.

Delebecque et al. created repeating RNA scaffolds that self-assemble in bacteria. Transcribed from plasmids in Escherichiacoli, these RNA scaffolds were used to facilitate flux through a metabolic pathway. The enzymes [FeFe] hydrogenase and ferrodoxin cata-lyse the reduction of protons to hydrogen. Fusing these proteins to RNA-binding domains allowed them to interact with RNA aptam-ers expressed in the same cells. Scaffolding was achieved by chain-ing the aptamers into repeating 2D units using a double-crossover motif; the scaffold-associated enzymes improved hydrogen output almost 50-fold122. This approach has recently been further refined and extended to increase the efficiency of pentadecane synthesis in E.coli123.

Bhadra and Ellington used products from in vitro transcrip-tion reactions to demonstrate dynamic RNA strand displace-ment cascades124. They employed transcribed RNA hairpins to construct circuits capable of cascading, amplification and logic. Although developed invitro, this demonstration the feasibility of using products of transcription for the construction of dynamic devices suggests that a similar approach may be used in the cell. Crossing into the realm of synthetic biology, Isaacsetal. engineered a class of riboregulators that rely on a strand-displacement mecha-nism for activation. Riboregulators inhibit bacterial mRNA transla-tion by hiding the ribosome binding site inside the stem of a hairpin loop; the repression can be relieved by expressing a short RNA that hybridizes to the loop domain and unfolds the stem structure125. These riboregulators have been further modified by Greenetal. to relieve many of the sequence constraints126.

Exogenously produced DNA devices have the advantage of closely mimicking those that have been developed for use invitro. On the other hand, by moving to systems that are transcribed within the cell, problems of delivery and expression become soluble using the more familiar tools of genetic engineering. As transcribed RNA nanotechnology develops, tools from more mature disciplines of genetic engineering will become increasingly valuable.

Recently, the possibility to reconcile these two approaches has emerged, thereby potentially allowing DNA systems to be expressed directly in cells rather than having to reinterpret them in a new nucleic acid substrate. The Lu group used a retron a bacteria-derived reverse transcriptase to express single-stranded DNA in bacteria. They used these DNA species to incrementally modify the specific regions of the bacterial chromosome, thus creating a popula-tion-level analog timer127. It is not much of a stretch to imagine that the same system could be further expanded to create components for DNA-based structures and dynamic systems.

OutlookDNA nanotechnology has made remarkable strides towards practi-cal applications in cellular settings. Nucleic acid structures in par-ticular have already quite successfully made the transition from the invitro to the invivo environment. Structures from tetrahedra to




origami have been shown to be stable in cells and can be readily modified into molecular transportation devices for siRNA, anti-bodies or small-molecule drugs. The most immediate application might be to use DNA nanostructures as programmable tools for interrogating cellular processes66. Even the use of nanostructures as multifunctional carriers for drug delivery seems to be within reach, with several groups already demonstrating functionality of nanostructure-based therapeutics in mouse models.

What challenges need to be met before DNA nanostructures can compete with alternative delivery technologies based on liposomes, polymers, aptamers and others58,85? First, we must develop a bet-ter understanding of the pharmacokinetics and biodistribution of DNA nanostructures. For example, one challenge is to design approaches that allow the selective uptake while inhibiting the non-selective uptake of DNA nanostructures by target cells; this is a common hurdle for any drug carrier, but is something the DNA nanotechnology field will need to work out. Additionally, potential immunostimulatory properties of DNA nanostructures must be investigated. Finally, production cost could be a concern, especially for large nanostructures such as DNA origami, which will prob-ably be at least as expensive to produce as antibodies ($300g1 or less128) or aptamers ($50g1, ref.58). Moreover, if the DNA nano-structures primarily serve as drug carriers, they will need to com-pete with polymer materials that can cost less than $1g1. However, alternative methods for synthesizing large amounts of high-quality DNA are being explored. For example, recently developed high-cell-density bioreactors can efficiently generate large quantities of M13 phage genome129. Combined with other approaches for producing high-quality short oligonucleotides in cells or enzymatically130,131, this technology provides a promising start towards developing low-cost production methods for DNA nanostructures in the future. Thus, it seems very likely that these challenges will be overcome, given the truly unique potential of DNA nanostructures to serve

as programmable, multifunctional, therapeutic systems that could eventually rival viruses in sophistication.

DNA-based therapeutics and diagnostics are set apart from more established approaches because of their capacity to respond to the surrounding environment. Molecular logic and conditional (un)hiding of drug moieties could decrease side effects and increase specificity. Even the relatively simple one- or two-input systems built so far have resulted in increased specificity and performance, and could be further improved with more complex multi-input logic. Diagnostic and therapeutic decisions are routinely based on the analysis of panels of multiple molecular markers, be they proteins, RNA, DNA, lipids, sugars or metabolites. For example, immunolo-gists must often consider large numbers of cell surface proteins to delineate all of the various cell types in a blood sample. Gene expres-sion classifiers that reliably distinguish different tissues and disease states are typically built on measurements of tens or hundreds of different RNA species. Given the success of dynamic DNA nano-technology in scaling up the size and reliability of molecular circuits in cell-free settings, it is intriguing to think that DNA biocomput-ers could eventually perform complex diagnostic tasks based on the analysis of tens of molecular markers directly in living organisms.

Beyond diagnostic and therapeutic devices, we could imagine synthetic DNA ecosystems that integrate motors, logic, structural elements and more to control and interrogate cellular behaviour in time and space. To realize such a vision and go beyond the delivery of mostly static structures, we still need to identify broadly appli-cable design principles that make it easy to translate any device that works reliably in cell-free settings to the cellular environment (Fig.6). New design strategies might include the delivery method, nucleic acid chemistry and sequence design, or even different reac-tion mechanisms. However, given the progress that has already been made, it is quite likely that DNA nanotechnology will become a use-ful complement to more traditional approaches for manipulating and controlling biological information.

Received 28 January 2015; accepted 29 July 2015; published online 3 September 2015

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AcknowledgementsWe would like to thank S.Douglas, N.Pierce, M.Schwarzkopf, S.Pun and D.Soloveichik for insightful comments and helpful feedback on the manuscript. This work was supported by NSF grants CCF-1317653and CAREER CBET-0954566.

Author contributionsAll authors contributed equally to this work.

Additional informationReprints and permissions information is available online at www.nature.com/reprints. Correspondence should be addressed to G.S.

Competing financial interestsThe authors declare no competing financial interests.



http://www.nature.com/reprints

DNA nanotechnology from the test tube to the cellCell-free DNA nanotechnologyDNA nanotechnology in lysates and fixed cellsFigure 1 | Applications of DNA nanotechnology at the interface with biology.Box 1 | Synthetic nucleic acids in the cellular environment.Figure 2 | In situ imaging of mRNA in fixed cells.Interacting with cell surface markersFigure 3 | Cell surface computation.DNA nanostructures as drug-delivery vehiclesDynamic DNA nanodevices inside living cellsFigure 4 | DNA nanomachines and logic gates in mammalian cells.Figure 5 | mRNA imaging in living cells.Genetically encoded structures and devicesOutlookReferencesFigure 6 | Complexity break for cellular DNA nanodevices?AcknowledgementsAuthor contributionsAdditional informationCompeting financial interests

DNA nanotechnology from the test tube to the cell

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