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Available online at www.sciencedirect.com
DNA-based assembly lines and nanofactories
Friedrich C Simmel
With the invention of the DNA origami technique, DNA selfassembly has reached a new level of sophistication. DNA can
now be used to arrange molecules and other nanoscale
components into almost arbitrary geometries — in two and
even three dimensions and with nanometer precision. One
exciting prospect is the realization of dynamic systems based
on DNA, in which chemical reactions are precisely controlled by
the spatial arrangement of components, ultimately resulting in
nanoscale analogs of molecular assembly lines or
‘nanofactories’. This review will discuss recent progress toward
this goal, ranging from DNA-templated synthesis over artificial
DNA-based enzyme cascades to first examples of ‘molecular
robots’.
Address
Lehrstuhl für Bioelektronik, Physik-Department der TU München,
Germany
Corresponding author: Simmel, Friedrich C ([email protected])
Current Opinion in Biotechnology 2012, 23:1–6
This review comes from a themed issue on
Nanobiotechnology
Edited by Fernando de la Cruz and Geoffrey Gadd
0958-1669/$ – see front matter
Published by Elsevier Ltd.
DOI 10.1016/j.copbio.2011.12.024
Introduction: DNA nanotechnology
Already in the early 1980s, Nadrian Seeman realized that
DNA might also be used in a non-biological context — as
a material for molecular ‘construction’ [1]. The basis of
DNA nanotechnology is the well-known fact that two
molecules of DNA pair up to form a double helix, when
their base sequences are complementary. DNA duplexes
are relatively rigid molecules on the nanoscale and hence
are used as molecular ‘beams’ for nanoconstruction
(Figure 1a). Linear assemblies are only of limited use,
and so a variety of branched DNA structures have been
developed for DNA nanotechnology. Many DNA ‘lattices’ are based on DNA four-way junctions, which are
derived from the biological ‘Holliday crossover’ structures. One of the milestones of the field was the creation
of extended DNA lattices from DNA ‘tiles’ that were
formed from two such crossover structures fused together
[2]. Later on, more complicated structures such as
‘crossbar tiles’ were developed that allowed for larger
assemblies (Figure 1b) [3,4]. DNA nanotechnology has
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significantly gained momentum since the demonstration
of the ‘origami’ technique by Rothemund in 2006 [5]. In
contrast to previous DNA assembly strategies that were
based on the association of many short oligonucleotides to
create a larger structure, DNA origami utilizes a long
single-stranded DNA ‘scaffold strand’ (Figure 1c). A large
number of rationally chosen ‘staple strands’ is added to
the scaffold and crosslinks it to fold into a specific molecular shape. This approach has turned out to be extraordinarily robust and efficient. Variations of the basic
origami principle have been later adopted for the construction of objects in 3D [6,7,8–10].
The specific mechanical properties of DNA molecules
together with ‘reversible’ hybridization reactions have
also been utilized for the construction of various
machine-like molecular assemblies [11] that can be controllably switched between several conformations or
‘states’. Such DNA-based switches are expected to find
application as autonomous biosensors in biomedicine, or
as actuators for DNA-based molecular motors and robots.
Using chemically modified DNA strands, DNA assemblies can be used to arrange nanoscale objects or molecules into well-defined geometries, with nanoscale
precision. One promising application is to use this capability for the precise control and optimization of chemical
reactions, and the realization of molecular ‘nanofactories’.
Reaction kinetics: proximity and geometry
In biology many chemical processes occur with extraordinary specificity and efficiency. One aspect that markedly
differentiates biological processes from conventional
chemistry ‘in a beaker’ is spatial organization and compartmentalization. Spatial organization may influence
reaction kinetics in a variety of ways. Holding two compounds in close proximity will increase the rate at which
they attempt to react with each other. This is important
for reactions with high activation barrier, or when the
reactants are only present at low concentrations and have
a low probability to meet in the first place. If in addition
one can control the orientation of reactants with respect to
each other, these may be arranged in an optimum geometry to facilitate bond formation.
A more complicated situation arises when reaction cascades with several consecutive steps and intermediate
compounds are considered. Such intermediates can be
unstable or toxic; they can diffuse away or react with other
species. Compartmentalization and spatial organization is
thought to prevent side-reactions and to increase local
concentrations of reactants and therefore improve the
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2 Nanobiotechnology
Figure 1
(a)
(b)
2 nm
(c)
17.6 nm
10.9 nm
Current Opinion in Biotechnology
DNA as a nanoscale building material. (a) DNA double helices are structurally rigid on the nanometer scale. (b) Many short DNA molecules can be
‘woven together’ to form supramolecular lattices. (c) In DNA origami, many short ‘staple strands’ (green/blue) hold together a long single-stranded
‘scaffold strand’ (gray). For clarity the double helical nature of DNA is not shown in detail here.
reaction flux through such cascades. In the context of
multi-enzyme complexes, these effects have been termed
‘metabolic channeling’ [12].
The effect of spatial arrangement strongly depends on the
physical process under consideration. Formation of
covalent bonds takes place on a sub-nm scale — this level
of control corresponds to the organization of ‘active
centers’ of many enzymes. Electron transfer reactions
that involve tunneling of electrons typically occur over
the distance of one or a few nanometers, energy transfer
between electric dipoles has a longer range and occurs on
a scale of several nanometers. For diffusive processes one
has to consider reactant concentrations and typical time
scales of a system. Quite generally, spatial arrangement
for reactions involving diffusion is only expected to play a
role for low concentrations and small diffusion coefficients — or in crowded environments [13].
DNA-directed synthesis
There are different levels of organization that DNA
nanotechnology may provide for the control of chemical
reactions: proximity, spatial order in one dimension, and
geometrical arrangement in two or even three dimensions. In DNA-directed synthesis, two DNA-linked reactants are brought into close proximity by hybridization to
a complementary template strand (Figure 2a). This
increases the effective concentration of the reagents
and therefore their reaction rate [14,15]. In principle,
DNA-directed synthesis can also be used to translate
a DNA sequence into artificial sequences composed
of other molecules. This has been employed for
sequence-controlled multistep synthesis [16] and also
for the realization of small-molecule libraries for combinatorial chemistry [17–21]. In DNA-templated synthesis,
the reactants are not held firmly in place — they simply
sample conformations and orientations by diffusion until
they react. This is somewhat controlled by the point of
attachment, the length and mechanical properties of the
linker through which the reactants are connected to the
DNA scaffold. The extreme specificity of many naturally
occurring enzymes arises from the precise spatial arrangement of the reactants to each other, which results from the
binding of substrates to the enzymes’ catalytic center. In
principle it should be possible to also construct ‘artificial
binding pockets’ using the three-dimensional origami
technique. Chemically modified DNA staple strands
could present functional groups that arrange and orient
reactants for a reaction within the pocket.
Reaction cascades and multi-enzyme
systems in biology
Many chemical systems in biology are organized on a
scale larger than the reactive centers of single enzymes,
for instance within multi-enzyme complexes. A variety of
such complexes are known to carry out intricate biosynthetic tasks, for which several catalytic steps have to be
performed ‘in series’, and the substrate is passed from one
catalytic site to the next without escaping to the bulk
phase. Such ‘channeled’ intermediates cannot participate
in competing reaction pathways, and they are present in a
higher local concentration than in the bulk. Arrangement
of enzymes tightly links the catalytic steps and therefore
also balances the flux through an enzyme cascade.
As a famous example, tryptophan synthase catalyzes the
formation of tryptophan from indole-3-glycerol phosphate in two catalytic steps. The two catalytic sites are
connected via an intramolecular tunnel of 2 nm length,
through which indole — the reaction intermediate — is
transferred [22,23]. Another example for multistep catalysis in a closed enzyme complex is found in fatty acid
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DNA-based assembly lines and nanofactories Simmel 3
Figure 2
(a)
A
B
A
B
(b)
Current Opinion in Biotechnology
Arranging chemical components with DNA molecules into close proximity allows for the control of chemical and enzymatic reactions. (a) Principle of
DNA-directed synthesis: DNA strands are modified with chemical moieties A and B. Hybridization to a complementary scaffold places A and B next to
each other and allows them to react. (b) The turnover of a multi-enzyme reaction can be enhanced by placing the enzymes into close proximity.
synthesis. Fungal fatty acid synthase, for example, is a
barrel-shaped reaction compartment, which contains all
the catalytic subunits required for the synthesis of fatty
acids [24]. While metabolic channeling undoubtedly
plays a role in such ‘closed’ complexes, it is less obvious
whether it is also important for enzymes that are simply
co-localized, and where intermediates can diffuse away
and mix with the bulk.
Apart from kinetic considerations, another benefit of
spatial arrangement of enzymes can be the concerted
action of several complementary enzymes to synergistically perform a complex chemical task. A well-known
example for this is the cellulosome — a huge enzyme
complex found in anaerobic bacteria that breaks down
plant cell walls [25].
Other examples of spatial organization are found in
signaling cascades, where molecular signals progress
along a chain of kinases that are aligned on scaffold
proteins [26]. Similar to the case of metabolic cascades,
the benefits of spatial organization are reduction of
crosstalk, context-dependent response, and speedy
transmission. Finally, another type of spatial positioning is found in photosynthetic light-harvesting and
reaction complexes, where efficient charge and energy
transfer reactions are achieved by the precise tuning of
distances and energies of chlorophylls and other molecules [27].
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Artificial enzyme cascades
The idea of artificially co-localizing enzymes to improve
reaction flux has already been attempted using a variety
of different approaches. Enzyme systems have been
immobilized on a variety of solid matrices, resulting in
an improved transfer of intermediate substrates under
certain conditions [28,29]. Other attempts were made to
enforce co-localization by chemically linking several
enzymes of interest to scaffolds, by recombinant engineering of fusion proteins [30], or by the utilization of
protein–protein interaction domains [31].
In the past decade, there have also been first attempts to
utilize DNA scaffolding for the creation of artificial multienzyme systems (Figure 2b). Niemeyer and coworkers
[32] first arranged NADH:FMN oxidoreductase and
luciferase onto double-stranded DNA scaffolds using
biotin–streptavidin linkages, later they also used enzymes
covalently linked to DNA to produce systems from glucose oxidase (GOX) and horseradish peroxidase (HRP)
[33]. In each case, an improved performance was observed
when the cooperating enzymes were held in proximity.
The first example of enzyme systems arranged on a larger
supramolecular DNA scaffold was demonstrated by Willner and coworkers [34]. Also using the GOX/HRP system, they reported a significant increase in overall
activity, when the two enzymes were placed in close
proximity. In addition, they also immobilized glucose
dehydrogenase and its tethered cofactor NAD+ on the
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4 Nanobiotechnology
Figure 3
(a)
(b)
(c)
Current Opinion in Biotechnology
DNA nanostructures such as DNA origami provide a higher level of spatial control than simple arrangements along linear scaffolds. (a) Components of
a more complex pathway can be arranged into close proximity on DNA origami (DNA double helices are represented as bars here). Choosing the
correct ‘stoichiometry’ on the platform may help to balance reaction fluxes. (b) To avoid diffusive loss of reaction intermediates to the bulk,
components may also be encapsulated into artificial reaction chambers (here a chamber made of a multi-helix bundle is indicated). (c) Many different
components can be brought together in an origami-based ‘nanofactory’. Here, molecular walkers transport components between two reaction centers
to prevent loss by diffusion. Other components such as nanoparticles are easily integrated into such assemblies.
scaffold, and observed different activities for different
tether lengths and distances. As could be expected,
researchers are now beginning to study enzymes
immobilized on origami structures [35]. Another impressive recent achievement in this context is the assembly of a
multi-enzyme complex along an aptamer-containing
RNA scaffold in Escherichia coli [36].
What advantages does DNA scaffolding have to offer over
approaches such as direct chemical linking of enzymes or
protein engineering? For one, DNA assembly is very
flexible — it is straightforward to systematically vary distances between enzymes, their order and stoichiometry
on a DNA scaffold. This could be used for ‘fast prototyping’ of enzyme cascades to find optimum configurations. One major strength of DNA-based assembly is its
potential to produce two-dimensional and even threedimensional arrangements. Rather than only placing
enzymes into close proximity, DNA could help to precisely define the geometry of multi-enzyme assemblies
(Figure 3a). For instance, a ‘donor’ enzyme could be
surrounded by other ‘acceptor’ enzymes to increase the
capture probability of diffusing intermediates, or one
could tether a substrate carrying ‘arm’ close to a row of
enzymes that consecutively modify the substrate. With
3D origami it should even be possible to mimic closed
enzyme assemblies such as fatty acid synthase, in which
all members of an enzyme-cascade reside within a supramolecular reaction chamber (Figure 3b).
An important advantage of enzyme cascades based on
fusion proteins is their use within metabolically engineered cells [30]. In contrast to chemical methods —
including DNA self-assembly — once a successful
multi-enzyme construct has been realized, its synthesis
can be easily scaled up for large-scale production using
biotechnological tools. With the further development of
in vivo RNA nanotechnology [36,37], however, the
benefits of the two different approaches may actually
be combined.
Controlling charge and energy transfer
Organization of molecules along a DNA scaffold may also
be used to control other physicochemical processes that
may play a role in future ‘nanofactories’. One of the
visions here is to mimic biological light-harvesting complexes and also photocatalytic energy conversion such as
in photosynthetic reaction centers. There are a large
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DNA-based assembly lines and nanofactories Simmel 5
number of publications on the organization of chromophores and energy transfer along linear DNA scaffolds
(e.g. [38,39]), and there have been many studies on charge
transport through DNA [40]. These studies have
been recently extended to more elaborate DNA nanoconstructs. For instance, Dwyer et al. studied energy
transfer between three chromophores immobilized on
a self-assembled DNA grid [41], while Tinnefeld and
coworkers demonstrated FRET cascades with four fluorophores on a planar DNA origami structure [42]. More
recently, Liu and coworkers reported on the fabrication
of a ‘light-harvesting system’ based on fluorophores
assembled around a DNA helix bundle [43].
Molecular robots and assembly lines
An exciting development of the past years are first
attempts to couple the mechanical motion of DNA-based
nanodevices to chemical synthesis. For instance, Chen
and Mao showed that mechanical switching of a DNA
nanodevice can be used to ‘choose’ between two alternative reactions [44]. More recently, He and Liu demonstrated how a DNA ‘walker’ autonomously performed
DNA-templated multistep synthesis by carrying a DNAlinked compound through a series of reactive ‘sites’
arranged along a track [45]. Their system worked faster
and more efficiently than previous DNA-directed multistep syntheses. Also using a DNA walking device, Gu
et al. constructed an origami-based ‘molecular assembly
line’ that could programmably collect different combinations of gold nanoparticles [46]. Their combination of
mechanical motion and programmable assembly already
very much resembles macroscale production lines, along
which programmable industrial robots put together parts
to manufacture a composite product. In this sense, these
recent developments are more ‘technomimetic’ than
‘biomimetic’.
Conclusions
In the past years, DNA has been proven to be an extraordinary material for the assembly of complex molecular
architectures. Moreover, DNA has also been shown to be
capable of directing chemical reactions resulting in
increased reaction efficiencies and a new approach for
combinatorial chemistry. Researchers have begun to
arrange enzymes into artificial reaction complexes, and
DNA nanomachines have been used to control chemical
synthesis and assembly processes. With the power of
DNA origami, much more complex systems seem to be
feasible in the future.
Probably the main strength of DNA-based assembly
is its flexibility in design. As indicated in this review,
DNA self-assembly could be utilized to integrate many
different components and physicochemical processes
within one system. For instance, multi-enzyme systems
could be arranged within artificial origami-based
reaction chambers, and their reactions could be coupled
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to photo-induced electron and energy-transfer processes,
which progress along artificial DNA-based antenna systems and electron transport chains. Programmable DNAbased motors and machines could then help to shuttle
molecular components between such reaction centers,
making the vision of a nanofactory quite seizable
(Figure 3c).
Potential applications of such ‘nanofactories’ can be
found in many different areas. Optimized enzymatic
reaction pathways might be used for the production of
novel drugs or biofuels, or for biodegradation. Integration
with sensor and computational functions may result in
‘autonomous’ nanodevices that are capable of contextdependent behavior, with possible applications in nanomedicine or in other complex, changing environments.
For practical use, many of these applications will require a
reduction in cost of DNA nanostructures and strategies
for scaling up their synthesis. This might be achieved by a
more extensive utilization of biotechnological tools, for
example, for in vivo production of nucleic acid scaffolds,
or by a standardization of origami ‘parts’.
Acknowledgements
The author acknowledges support through the DFG Cluster of Excellence
Nanosystems Initiative Munich (NIM).
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