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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
AND
a b
c d
Cell
d
Smart therapeutics Drug delivery
Imaging Cell biology
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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750 NATURE NANOTECHNOLOGY | VOL 10 | SEPTEMBER 2015 |
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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
b
amRNA target
Wild-type probe
Wild-typeRNA target
Guide probe Guide probe
Mutant RNARNA target
Mutant probe
l1
l2
H1
H2
Wild-type RNAMutant RNA
Unclassified RNA
Mutant detectionprobe
SNV detection
Heterozygotic cell
1 2
3 4
50 m
5 m
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.
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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
AND0
00
Cell
1
00
Cell
0
10
Cell
1
11
Cell
AND AND AND
DNA circuits
Non-target cell
Target cell
Label Evaluate Report
Initiator
ReporterQF
QF
Q
Q
F
F
Negative
Positive
a
b
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.
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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.
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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
a
bAND logic
TGN
Substrate
Fu-IFu Tf-ITf TF receptor
AND gate
miR21miR125b
Input AInput B
Salt
Salt OH
H+
Salt
Salt OH
H+
LE
RE
SE
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.
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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
cControl
+
20 m
Target mRNA
mRNA 3 UTR target
-actin mRNA Scrambled probe Merge
mRNA
1 2 3 1 2 3
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.
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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
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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
References1. Bloomfield, V.A., Crothers, D.M. & Ignacio
Tinoco, J. Nucleic Acids:
Structures, Properties and Functions (University Science Books,
2000).2. SantaLucia, J. & Hicks, D. The thermodynamics of DNA
structural motifs.
Annu. Rev. Biophys. Biomol. Struct. 33, 415440 (2004).3.
Carlson, R. The changing economics of DNA synthesis. Nature
Biotechnol.
27,10911094 (2009).4. Dittmer, W.U., Reuter, A. & Simmel,
F.C. A. DNA-based machine
that can cyclically bind and release thrombin. Angew. Chem. Int.
Ed. 43, 35503553 (2004).
5. Yurke, B., Mills, A.P. Jr & Cheng, S.L. DNA
implementation of addition in which the input strands are separate
from the operator strands. BioSystems 52,165174 (1999).
6. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro,
E. An autonomous molecular computer for logical control of gene
expression. Nature 429,423429 (2004).
7. Ko, S., Liu, H., Chen, Y. & Mao, C. DNA nanotubes as
combinatorial vehicles for cellular delivery. Biomacromolecules 9,
30393043 (2008).
Cellular uptake of large DNA nanostructures was first
demonstrated in thiswork.
8. Seeman, N.C. Nucleic acid junctions and lattices. J.Theor.
Biol. 99, 237247 (1982).
9. Kallenbach, N.R., Ma, R.-I. & Seeman, N.C. An immobile
nucleic acid junction constructed from oligonucleotides. Nature
305, 829831 (1983).
10. Chen, J. & Seeman, N.C. Synthesis from DNA of a molecule
with the connectivity of a cube. Nature 350, 631633 (1991).
11. Winfree, E., Liu, F., Wenzler, L.A. & Seeman, N.C.
Design and self-assembly of two-dimensional DNA crystals. Nature
394, 539544 (1998).
12. Goodman, R.P. et al. Rapid chiral assembly of rigid DNA
building blocks for molecular nanofabrication. Science 310,
16611665 (2005).
13. Zheng, J. et al. From molecular to macroscopic via the
rational design of a self-assembled 3D DNA crystal. Nature 461,
7477 (2009).
2003 2006
6
2734 35
39
In vitro circuitsIn vivo circuits 37
41 96106
38
60 107?
5997113114
2009Year of publication
Ope
ratio
ns
2012 20150
10
20
30
40
50
60
70
Figure 6 | Complexity break for cellular DNA nanodevices? The
complexity of cell-free DNA logic circuits and similar dynamic
devices has increased by almost two orders of magnitude over the
past decade. In cellular settings, dynamic devices with only two or
three independent operations have so far been demonstrated. This
suggests that design principles for adapting dynamic DNA
nanodevices to cells are yet to be uncovered. Each coloured dot and
number represent a specific reaction network and associated
publication (reference number); trend lines are included to guide
the eye. An operation is defined as a unique (sequence-specific)
connection, such as a strand displacement reaction or DNAzyme
cleavage event within a network. A circuit with n gates arranged in
a cascade is considered to be equally complex as a circuit with n
independent gates operating in parallel, even though the latter is
probably easier to realize experimentally. Moreover, multi-turnover
catalytic reactions are weighed equally against single-step
reactions, which potentially underestimates the complexity of the
former.
REVIEW ARTICLENATURE NANOTECHNOLOGY DOI:
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2015 Macmillan Publishers Limited. All rights reserved
-
758 NATURE NANOTECHNOLOGY | VOL 10 | SEPTEMBER 2015 |
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14. Shih, W.M., Quispe, J.D. & Joyce, G.F. A 1.7-kilobase
single-stranded DNA that folds into a nanoscale octahedron. Nature
427, 618621 (2004).
15. Yan, H., LaBean, T.H., Feng, L. & Reif, J.H. Directed
nucleation assembly of DNA tile complexes for barcode-patterned
lattices. Proc. Natl Acad. Sci. USA 100, 81038108 (2003).
16. Schulman, R. & Winfree, E. Synthesis of crystals with a
programmable kinetic barrier to nucleation. Proc. Natl Acad. Sci.
USA 104, 1523615241 (2007).
17. Rothemund, P.W. K., Papadakis, N. & Winfree, E.
Algorithmic self-assembly of DNA Sierpinski triangles. PLoS Biol.
2, e424 (2004).
18. He, Y. et al. Hierarchical self-assembly of DNA into
symmetric supramolecular polyhedra. Nature 452, 198201 (2008).
19. Yan, H., Park, S.H., Finkelstein, G., Reif, J.H. &
LaBean, T.H. DNA-templated self-assembly of protein arrays and
highly conductive nanowires. Science 301,18821884 (2003).
20. Rothemund, P.W. K. Folding DNA to create nanoscale shapes
and patterns. Nature 440, 297302 (2006).
21. Douglas, S.M. et al. Self-assembly of DNA into nanoscale
three-dimensional shapes. Nature 459, 414418 (2009).
22. Ke, Y. et al. Scaffolded DNA origami of a DNA tetrahedron
molecular container. Nano Lett. 9, 24452447 (2009).
23. Andersen, E.S. et al. Self-assembly of a nanoscale DNA box
with a controllable lid. Nature 459, 7376 (2009).
24. Dietz, H., Douglas, S.M. & Shih, W.M. Folding DNA into
twisted and curved nanoscale shapes. Science 325, 725730
(2009).
25. Adleman, L.M. Molecular computation of solutions to
combinatorial problems. Science 266, 10211024 (1994).
26. Yurke, B., Turberfield, A.J., Mills, A.P. Jr, Simmel, F.C.
& Neumann, J.L. ADNA-fuelled molecular machine made of DNA.
Nature 406, 605608 (2000).
27. Seelig, G., Soloveichik, D., Zhang, D.Y. & Winfree, E.
Enzyme-free nucleic acid logic circuits. Science 314, 15851588
(2006).
28. Dirks, R.M. & Pierce, N.A. Triggered amplification by
hybridization chain reaction. Proc. Natl Acad. Sci. USA 101,
1527515278 (2004).
29. Kay, E.R., Leigh, D. A & Zerbetto, F. Synthetic
molecular motors and mechanical machines. Angew. Chem. Int. Ed. 46,
72191 (2007).
30. Bath, J. & Turberfield, A.J. DNA nanomachines. Nature
Nanotech. 2, 274284 (2007).
31. Omabegho, T., Sha, R. & Seeman, N.C. A bipedal DNA
Brownian motor with coordinated legs. Science 324, 6771 (2009).
32. Lund, K. et al. Molecular robots guided by prescriptive
landscapes. Nature 465, 206210 (2010).
33. Muscat, R.A., Bath, J. & Turberfield, A.J. A
programmable molecular robot. Nano Lett. 11, 982987 (2011).
34. Wickham, S.F. J. et al. A DNA-based molecular motor that can
navigate a network of tracks. Nature Nanotech. 7, 169173
(2012).
35. Chen, Y.-J. et al. Programmable chemical controllers made
from DNA. NatureNanotech. 8, 755762 (2013).
36. Qian, L., Winfree, E. & Bruck, J. Neural network
computation with DNA strand displacement cascades. Nature 475,
368372 (2011).
37. Qian, L. & Winfree, E. Scaling up digital circuit
computation with DNA strand displacement cascades. Science 332,
11961201 (2011).
38. Elbaz, J. et al. DNA computing circuits using libraries of
DNAzyme subunits. Nature Nanotech. 5, 417422 (2010).
39. Pei, R., Matamoros, E., Liu, M., Stefanovic, D. &
Stojanovic, M.N. Training a molecular automaton to play a game.
Nature Nanotech. 5, 773777 (2010).
40. Seelig, G., Yurke, B. & Winfree, E. Catalyzed relaxation
of a metastable DNA fuel. J.Am. Chem. Soc. 128, 1221112220
(2006).
41. Zhang, D.Y., Turberfield, A.J., Yurke, B. & Winfree, E.
Engineering entropy-driven reactions and networks catalyzed by DNA.
Science 318, 11211125 (2007).
42. Zhang, D.Y. & Winfree, E. Dynamic allosteric control of
noncovalent DNA catalysis reactions. J.Am. Chem. Soc. 130,
1392113926 (2008).
43. Turberfield, A.J. et al. DNA fuel for free-running
nanomachines. Phys. Rev. Lett. 90, 118102 (2003).
44. Bois, J.S. et al. Topological constraints in nucleic acid
hybridization kinetics. Nucleic Acids Res. 33, 40904095 (2005).
45. Benenson, Y. et al. Programmable and autonomous computing
machine made of biomolecules. Nature 414, 430434 (2001).
46. Mei, Q. et al. Stability of DNA origami nanoarrays in cell
lysate. Nano Lett. 11,14771482 (2011).
47. Conway, J.W., McLaughlin, C.K., Castor, K.J. & Sleiman,
H. DNA nanostructure serum stability: greater than the sum of its
parts. Chem.Commun. 49, 11721174 (2013).
48. Hahn, J., Wickham, S.F. J., Shih, W.M. & Perrault, S.D.
Addressing the instability of DNA nanostructures in tissue culture.
ACS Nano 8, 87658775 (2014).
49. Keum, J.-W. & Bermudez, H. Enhanced resistance of DNA
nanostructures to enzymatic digestion. Chem. Commun. 70367038
(2009).
50. Castro, C.E. et al. A primer to scaffolded DNA origami.
Nature Methods 8,221229 (2011).
51. Choi, H.M. T. et al. Programmable in situ amplification for
multiplexed imaging of mRNA expression. Nature Biotechnol. 28,
12081212 (2010).
52. Choi, H.M. T., Beck, V.A. & Pierce, N.A. Next-generation
in situ hybridization chain reaction: higher gain, lower cost,
greater durability. ACSNano 8, 42844294 (2014).
53. Levesque, M.J., Ginart, P., Wei, Y. & Raj, A.
Visualizing SNVs to quantify allele-specific expression in single
cells. Nature Methods 10, 865867 (2013).
Taking advantage of the specificity of toehold-mediated strand
displacement reactions, this work demonstrated that
single-nucleotide variants on single RNA transcripts can be
detected using smFISH-based imaging probes.
54. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden,
A. & Tyagi, S. Imaging individual mRNA molecules using multiple
singly labeled probes. Nature Methods 5, 877879 (2008).
55. Duose, D.Y. et al. Configuring robust DNA strand
displacement reactions for in situ molecular analyses. Nucleic
Acids Res. 40, 32893298 (2012).
56. Jungmann, R. et al. Single-molecule kinetics and
super-resolution microscopy by fluorescence imaging of transient
binding on DNA origami. Nano Lett. 10,47564761 (2010).
57. Jungmann, R. et al. Multiplexed 3D cellular super-resolution
imaging with DNA-PAINT and Exchange-PAINT. Nature Methods 11,
313318 (2014).
58. Keefe, A., Pai, S. & Ellington, A. Aptamers as
therapeutics. Nature Rev. Drug Discov. 9, 537550 (2010).
59. Rudchenko, M. et al. Autonomous molecular cascades for
evaluation of cell surfaces. Nature Nanotech. 8, 580586 (2013).
This work successfully used strand displacement cascades to
classify different cell types, thereby demonstrating a scalable
approach for the analysis of cellular information.
60. Douglas, S.M., Bachelet, I. & Church, G.M. A logic-gated
nanorobot for targeted transport of molecular payloads. Science
335, 831834 (2012).
Proof-of-principle demonstration of a novel class of conditional
therapeutics that combine protective DNA origami structures with
molecular logic.
61. Tuerk, C. & Gold, L. Systematic evolution of ligands by
exponential enrichment: RNA ligands to bacteriophage T4 DNA
polymerase. Science 249,505510 (1990).
62. Ellington, A.D. & Szostak, J. In vitro selection of RNA
molecules that bind specific ligands. Nature 346, 818822
(1990).
63. Amir, Y. et al. Universal computing by DNA origami robots in
a living animal. Nature Nanotech. 9, 353357 (2014).
64. You, M. et al. DNA nano-claw: logic-based autonomous cancer
targeting and therapy. J.Am. Chem. Soc. 136, 12561259 (2014).
65. You, M., Zhu, G., Chen, T., Donovan, M.J. & Tan, W.
Programmable and multiparameter DNA-based logic platform for cancer
recognition and targeted therapy. J.Am. Chem. Soc. 137, 667674
(2015).
66. Shaw, A. et al. Spatial control of membrane receptor
function using ligand nanocalipers. Nature Methods 11, 841846
(2014).
By showing that cells are sensitive to the spatial organization
of protein ligands arranged on a DNA origami, the authors provide
an intriguing example of the use of nanostructures as tools for
cell biology.
67. Chandra, R.A., Douglas, E.S., Mathies, R.A., Bertozzi, C.R.
& Francis,M.B. Programmable cell adhesion encoded by DNA
hybridization. Angew. Chem. Int. Ed. 45, 896901 (2006).
68. Saxon, E. & Bertozzi, C.R. Cell surface engineering by a
modified Staudinger reaction. Science 287, 20072010 (2000).
69. Gartner, Z.J. & Bertozzi, C.R. Programmed assembly of
3-dimensional microtissues with defined cellular connectivity.
Proc. Natl Acad. Sci. USA 106,46064610 (2009).
This work demonstrated a novel strategy for the bottom-up
construction of microtissues using DNA sequence-programmed
connectivity.
70. Liu, J.S., Farlow, J.T., Paulson, A.K., Labarge, M.A. &
Gartner, Z.J. Programmed cell-to-cell variability in Ras activity
triggers emergent behaviors during mammary epithelial
morphogenesis. Cell Rep. 2, 14611470 (2012).
71. Langecker, M. et al. Synthetic lipid membrane channels
formed by designed DNA nanostructures. Science 338, 932936
(2012).
72. Burns, J.R. et al. Lipid-bilayer-spanning DNA nanopores with
a bifunctional porphyrin anchor. Angew. Chem. Int. Ed. 52,
1206912072 (2013).
73. Burns, J.R., Al-Juffali, N., Janes, S.M. & Howorka, S.
Membrane-spanning DNA nanopores with cytotoxic effect. Angew. Chem.
Int. Ed. 53, 1246612470 (2014).
74. Walsh, A.S. et al. DNA cage delivery to mammalian cells. ACS
Nano 5, 54275432 (2011).
75. Schller, V.J. et al. Cellular immunostimulation by
CpG-sequence-coated DNA origami structures. ACS Nano 5, 96969702
(2011).
REVIEW ARTICLE NATURE NANOTECHNOLOGY DOI:
10.1038/NNANO.2015.195
2015 Macmillan Publishers Limited. All rights reserved
-
NATURE NANOTECHNOLOGY | VOL 10 | SEPTEMBER 2015 |
www.nature.com/naturenanotechnology 759
76. Liang, L. et al. Single-particle tracking and modulation of
cell entry pathways of a tetrahedral DNA nanostructure in live
cells. Angew. Chem. Int. Ed. 53, 77457750 (2014).
77. Mikkil, J. et al. Virus-encapsulated DNA origami
nanostructures for cellular delivery. Nano Lett. 14, 21962200
(2014).
78. Perrault, S.D. & Shih, W.M. Virus-inspired membrane
encapsulation of DNA nanostructures to achieve in vivo stability.
ACS Nano 8, 51325140 (2014).
The authors showed that lipid encapsulation of DNA octahedrons
results in a reduced immune response and greatly enhanced
bioavailability in circulation in mouse models.
79. Hemmi, H. et al. A Toll-like receptor recognizes bacterial
DNA. Nature 408, 740745 (2000).
80. Nishikawa, M., Matono, M., Rattanakiat, S., Matsuoka, N.
& Takakura, Y. Enhanced immunostimulatory activity of
oligodeoxynucleotides by Y-shape formation. Immunology 124, 247255
(2008).
The first demonstration of drug delivery using DNA
nanostructures; Y-shaped DNA nanostructures decorated with CpG
motifs were used to trigger immune responses in living cells.
81. Rattanakiat, S., Nishikawa, M., Funabashi, H., Luo, D. &
Takakura, Y. The assembly of a short linear natural
cytosine-phosphate-guanine DNA into dendritic structures and its
effect on immunostimulatory activity. Biomaterials 30, 57015706
(2009).
82. Mohri, K. et al. Design and development of nanosized DNA
assemblies in polypod-like structures as efficient vehicles for
immunostimulatory cpg motifs to immune cells. ACS Nano 6, 59315940
(2012).
83. Li, J. et al. Self-assembled multivalent DNA nanostructures
for noninvasive intracellular delivery of immunostimulatory CpG
oligonucleotides. ACS Nano 5,87838789 (2011).
84. Liu, X. et al. A DNA nanostructure platform for directed
assembly of synthetic vaccines. Nano Lett. 12, 42544259 (2012).
85. Davis, M.E., Chen, Z. (Georgia) & Shin, D.M.
Nanoparticle therapeutics: an emerging treatment modality for
cancer. Nature Rev. Drug Discov. 7, 771782 (2008).
86. Chang, M., Yang, C.-S. & Huang, D.-M. Aptamer-conjugated
DNA icosahedral nanoparticles as a carrier of doxorubicin for
cancer therapy. ACS Nano 5,61566163 (2011).
87. Jiang, Q. et al. DNA origami as a carrier for circumvention
of drug resistance. J.Am. Chem. Soc. 134, 1339613403 (2012).
88. Kim, K.-R. et al. Drug delivery by a self-assembled DNA
tetrahedron for overcoming drug resistance in breast cancer cells.
Chem. Commun. 49, 20102012 (2013).
89. Zhao, Y.-X. et al. DNA origami delivery system for cancer
therapy with tunable release properties. ACS Nano 6, 86848691
(2012).
90. Zhu, G. et al. Self-assembled, aptamer-tethered DNA
nanotrains for targeted transport of molecular drugs in cancer
theranostics. Proc. Natl Acad. Sci. USA 110, 79988003 (2013).
91. Zhang, Q. et al. DNA origami as an in vivo drug delivery
vehicle for cancer therapy. ACS Nano 8, 66336643 (2014).
92. Keum, J.W., Ahn, J.H. & Bermudez, H. Design, assembly,
and activity of antisense DNA nanostructures. Small 7, 35293535
(2011).
93. Lee, H. et al. Molecularly self-assembled nucleic acid
nanoparticles for targeted invivo siRNA delivery. Nature Nanotech.
7, 389393 (2012).
94. Chen, G. et al. Enzymatic synthesis of periodic DNA
nanoribbons for intracellular pH sensing and gene silencing. J.Am.
Chem. Soc. 137, 38443851 (2015).
95. Pei, H. et al. Reconfigurable three-dimensional DNA
nanostructures for the construction of intracellular logic sensors.
Angew. Chem. Int. Ed. 51, 90209024 (2012).
96. Modi, S. et al. A DNA nanomachine that maps spatial and
temporal pH changes inside living cells. Nature Nanotech. 4, 325330
(2009).
97. Modi, S., Nizak, C., Surana, S., Halder, S. & Krishnan,
Y. Two DNA nanomachines map pH changes along intersecting endocytic
pathways inside the same cell. Nature Nanotech. 8, 459467
(2013).
98. Tyagi, S. & Kramer, F.R. Molecular beacons: probes that
fluoresce upon hybridization. Nature Biotechnol. 14, 303308
(1996).
99. Chen, A.K., Davydenko, O., Behlke, M.A. & Tsourkas, A.
Ratiometric bimolecular beacons for the sensitive detection of RNA
in single living cells. Nucleic Acids Res. 38, e148 (2010).
100. Mhlanga, M.M., Vargas, D.Y., Fung, C.W., Kramer, F.R. &
Tyagi, S. tRNA-linked molecular beacons for imaging mRNAs in the
cytoplasm of living cells. Nucleic Acids Res. 33, 19021912
(2005).
101. Zhang, X., Song, Y., Shah, A. & Lekova, V. Quantitative
assessment of ratiometric bimolecular beacons as a tool for imaging
single engineered RNA transcripts and measuring gene expression in
living cells. Nucleic Acids Res. 41, e152 (2013).
102. Bratu, D.P., Cha, B.-J., Mhlanga, M.M., Kramer, F.R. &
Tyagi, S. Visualizing the distribution and transport of mRNAs in
living cells. Proc. Natl Acad. Sci. USA 100, 1330813313 (2003).
103. Santangelo, P.J. et al. Single moleculesensitive probes for
imaging RNA in live cells. Nature Methods 6, 347349 (2009).
104. Rosi, N.L. et al. Oligonucleotide-modified gold
nanoparticles for intracellular gene regulation. Science 312,
10271030 (2006).
Nanoflares provided the first example of strand displacement
reactions with an endogenous RNA input inside living cells.
105. Alhasan, A.H., Patel, P.C., Choi, C.H. J. & Mirkin,
C.A. Exosome encased spherical nucleic acid gold nanoparticle
conjugates as potent microRNA regulation agents. Small 10, 186192
(2014).
106. Prigodich, A.E. et al. Nano-flares for mRNA regulation and
detection. ACSNano 3, 21472152 (2009).
107. Halo, T.L. et al. NanoFlares for the detection, isolation,
and culture of live tumor cells from human blood. Proc. Natl Acad.
Sci. USA 111, 1710417109 (2014).
108. Afonin, K.A. et al. Activation of different split
functionalities on re-association of RNADNA hybrids. Nature
Nanotech. 8, 296304 (2013).
109. Chen, S.X., Zhang, D.Y. & Seelig, G. Conditionally
fluorescent molecular probes for detecting single base changes in
double-stranded DNA. NatureChem. 5, 782789 (2013).
110. Xie, Z., Liu, S.J., Bleris, L. & Benenson, Y. Logic
integration of mRNA signals by an RNAi-based molecular computer.
Nucleic Acids Res. 38, 26922701 (2010).
111. Hochrein, L.M., Schwarzkopf, M., Shahgholi, M., Yin, P.
& Pierce, N.A. Conditional dicer substrate formation via shape
and sequence transduction with small conditional RNAs. J.Am. Chem.
Soc. 135, 1732217330 (2013).
112. Kumar, D., Kim, S.H. & Yokobayashi, Y. Combinatorially
inducible RNA interference triggered by chemically modified
oligonucleotides. J.Am. Chem. Soc. 133, 27832788 (2011).
113. Kahan-Hanum, M., Douek, Y., Adar, R. & Shapiro, E. A
library of programmable DNAzymes that operate in a cellular
environment. Sci. Rep. 3,1535 (2013).
114. Hemphill, J. & Deiters, A. DNA computation in mammalian
cells: microRNA logic operations. J.Am. Chem. Soc. 135, 1051210518
(2013).
115. Yu, J., Liu, Z., Jiang, W., Wang, G. & Mao, C. De novo
design of an RNA tile that self-assembles into a homo-octameric
nanoprism. Nature Commun. 6, 16 (2015).
116. Lee, J.B., Hong, J., Bonner, D.K., Poon, Z. & Hammond,
P.T. Self-assembled RNA interference microsponges for efficient
siRNA delivery. Nature Mater. 11,316322 (2012).
117. Severcan, I. et al. A polyhedron made of tRNAs. Nature
Chem. 2, 772779 (2010).
118. Ohno, H. et al. Synthetic RNA-protein complex shaped like
an equilateral triangle. Nature Nanotech. 6, 116120 (2011).
119. Chworos, A. et al. Building programmable jigsaw puzzles
with RNA. Science 306, 20682073 (2004).
120. Afonin, K. A. et al. In vitro assembly of cubic RNA-based
scaffolds designed in silico. Nature Nanotechnol. 5, 676682
(2010).
121. Geary, C., Rothemund, P.W. K. & Andersen, E.S. A
single-stranded architecture for cotranscriptional folding of RNA
nanostructures. Science 345,799804 (2014).
122. Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye,
F.A. Organization of intracellular reactions with rationally
designed RNA assemblies. Science 333,470474 (2011).
This work used self-assembled RNA scaffolds to increase the
efficiency of hydrogen production in bacteria, thus demonstrating
the functional use of RNA architectures in vivo.
123. Sachdeva, G., Garg, A., Godding, D., Way, J.C. &
Silver, P. A. In vivo co-localization of enzymes on RNA scaffolds
increases metabolic production in a geometrically dependent manner.
Nucleic Acids Res. 42, 94939503 (2014).
124. Bhadra, S. & Ellington, A.D. Design and application of
cotranscriptional non-enzymatic RNA circuits and signal
transducers. Nucleic Acids Res. 42, e58 (2014).
125. Isaacs, F.J. et al. Engineered riboregulators enable
post-transcriptional control of gene expression. Nature Biotechnol.
22, 841847 (2004).
126. Green, A.A., Silver, P.A., Collins, J.J. & Yin, P.
Toehold switches: de-novo-designed regulators of gene expression.
Cell 159, 925939 (2014).
127. Farzadfard, F. & Lu, T.K. Genomically encoded analog
memory with precise invivo DNA writing in living cell populations.
Science 346, 6211 (2014).
128. Kelley, B. Industrialization of mAb production technology:
the bioprocessing industry at a crossroads. MAbs 1, 440449
(2009).
129. Kick, B., Praetorius, F., Dietz, H. & Weuster-Botz, D.
Efficient production of single-stranded phage DNA as scaffolds for
DNA origami. Nano Lett. 15,46724676 (2015).
130. Ducani, C., Kaul, C., Moche, M., Shih, W.M. & Hgberg,
B. Enzymatic production of monoclonal stoichiometric
single-stranded DNA oligonucleotides. Nature Methods 10, 647652
(2013).
131. Gu, H. & Breaker, R.R. Production of single-stranded
DNAs by self-cleavage of rolling-circle amplification products.
Biotechniques 54, 337343 (2013).
REVIEW ARTICLENATURE NANOTECHNOLOGY DOI:
10.1038/NNANO.2015.195
2015 Macmillan Publishers Limited. All rights reserved
-
760 NATURE NANOTECHNOLOGY | VOL 10 | SEPTEMBER 2015 |
www.nature.com/naturenanotechnology
132. Gilleron, J. et al. Image-based analysis of lipid
nanoparticlemediated siRNA delivery, intracellular trafficking and
endosomal escape. Nature Biotechnol. 31, 638646 (2013).
133. Sahay, G. et al. Efficiency of siRNA delivery by lipid
nanoparticles is limited by endocytic recycling. Nature Biotechnol.
31, 653658 (2013).
134. Bao, G., Rhee, W.J. & Tsourkas, A. Fluorescent probes
for live-cell RNA detection. Annu. Rev. Biomed. Eng. 11, 2547
(2009).
135. Fisher, T.L., Terhorst, T., Cao, X. & Wagner, R.W.
Intracellular disposition and metabolism of fluorescently-labeled
unmodified and modified oligonucleotides microinjected into
mammalian cells. Nucleic Acids Res. 21,38573865 (1993).
136. Watts, J.K., Deleavey, G.F. & Damha, M.J. Chemically
modified siRNA: tools and applications. Drug Discov. Today 13,
842855 (2008).
137. Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H.
Tolerance for mutations and chemical modifications in a siRNA.
Nucleic Acids Res. 31, 589595 (2003).
138. Bramsen, J.B. et al. A large-scale chemical modification
screen identifies design rules to generate siRNAs with high
activity, high stability and low toxicity. Nucleic Acids Res. 37,
28672881 (2009).
139. Lukacs, G.L. et al. Size-dependent DNA mobility in
cytoplasm and nucleus. J.Biol. Chem. 275, 16251629 (2000).
140. Schoen, I., Krammer, H. & Braun, D. Hybridization
kinetics is different inside cells. Proc. Natl Acad. Sci. USA 106,
2164921654 (2009).
141. Manche, L., Green, S.R., Schmedt, C. & Mathews, M.B.
Interactions between double-stranded RNA regulators and the protein
kinase DAI. Mol. Cell. Biol. 12, 52385248 (1992).
142. Krieg, A.M. Therapeutic potential of Toll-like receptor 9
activation. NatureRev. Drug Discov. 5, 471484 (2006).
143. Shir, A. & Levitzki, A. Inhibition of glioma growth by
tumor-specific activation of double-stranded RNA-dependent protein
kinase PKR. NatureBiotechnol. 20, 895900 (2002).
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.
REVIEW ARTICLE NATURE NANOTECHNOLOGY DOI:
10.1038/NNANO.2015.195
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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