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This is an electronic reprint of the original article.This reprint may differ from the original in pagination and typographic detail.
Author(s): Linko, Veikko & Kostiainen, Mauri A.
Title: Automated design of DNA origami
Year: 2016
Version: Post print
Please cite the original version:Linko, Veikko & Kostiainen, Mauri A. 2016. Automated design of DNA origami. NatureBiotechnology. Volume 34, Issue 8. 826-827. ISSN 1087-0156 (printed). DOI:10.1038/nbt.3647.
Rights: © 2016 Springer Nature. This is the post print version of the following article: Linko, Veikko & Kostiainen,Mauri A. 2016. Automated design of DNA origami. Nature Biotechnology. Volume 34, Issue 8. 826-827.ISSN 1087-0156 (printed). DOI: 10.1038/nbt.3647, which has been published in final form athttp://www.nature.com/nbt/journal/v34/n8/full/nbt.3647.html.
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Automated design of DNA origami
Veikko Linko & Mauri A. Kostiainen
A top-down computational method enables fabrication of DNA objects in any shape.
The past decade has witnessed remarkable advances in techniques for assembling DNA molecules
into nanoscale, three-dimensional shapes through programmed Watson-Crick base pairing. The
availability of design software of increasing modularity and power has simplified the creation of
complex DNA architectures. Despite this progress, in silico design of DNA nanostructures remains
a challenge, and all but a few experienced research groups have the expertise to fully implement the
methods in practice. A new study1 ingeniously tackles these issues by extending a recently
developed approach that proceeds not from the bottom up but from the top down, beginning with a
simple surface representation of the desired three-dimensional DNA shape. Writing in Science,
Bathe and colleagues1 introduce a top-down strategy that is fully automated, requiring little manual
intervention, and that can produce any desired shape. The method is likely to open up the field to a
broader cadre of researchers and to facilitate practical applications of these nanostructures in
diverse areas of biotechnology.
The assembly of DNA nanostructures with high spatial addressability has been
dubbed ‘DNA origami’ because of its affinity with the traditional Japanese art of paper folding2. In
the most widely used method, a single-stranded DNA ‘scaffold’ of ~7000 nucleotides in length is
folded into a predetermined shape using dozens of short, single-stranded ‘staples’ that are unique in
sequence. In a nutshell, an origami shape is constructed in a thermal annealing procedure as the
staple strands hybridize with multiple scaffold segments and form double-helical domains (i.e., the
short strands ‘staple’ the segments together). A correct tertiary structure is then formed according to
the selected architectural rules—the interconnected double-helical secondary structures adopt
spatial orientations that are encoded by the strand crossover positions in the design.
With improvements to the original origami method and the invention of alternative
design techniques, the space of possible DNA shapes has expanded considerably3. These now
include custom multilayer folds, structures with twists and bends, scaffold-free ‘LEGO-like’
designs and ‘wireframe’-based meshed constructions in which DNA edges are employed to connect
the vertices of the 3D object3,4. Underlying the increasing complexity of shapes are ever-more
potent computational tools for designing and analyzing such objects. For example, researchers
routinely use the design software caDNAno and the finite-element-based simulation software
CanDo for prototyping and predicting the shape of multilayer DNA structures in honeycomb and
square lattice geometries5. In addition, computational frameworks for making lattice-free DNA
structures are available6.
To take advantage of the above-mentioned methods, however, both the routing of the
scaffold strand and the design of the staple strands have to be performed manually, which often
requires basic knowledge of the structural properties and possible folding paths of DNA molecules.
Thus, the user defines the DNA sequences that will eventually form the desired object in a process
that requires considerable human intervention.
This conventional design strategy was turned on its head last year by the first top-
down software for building wireframe DNA nanoarchitectures7. The software vHelix7 is based on
selecting a target shape that fits to the length of the available scaffold and subsequently creating a
polyhedral meshwork of the object—an approach well known in computer graphics. The algorithm
routes the scaffold along the Eulerian meshwork and adds the staple strands to the design. Before
the actual ‘staple strand’ sequences are computed, a physical model is generated to understand
whether there is undesirable steric strain in the structure that requires relaxation. The authors
showed the feasibility of their semi-automated ‘3D printing’ technique by producing several
fascinating DNA structures with spherical topologies, such as the nanosized ‘Stanford bunny’.
Recently, the same laboratory extended their method by creating two-dimensional DNA shapes
derived from the flat-sheet triangulated meshes8.
Now, in the next step, Bathe and colleagues1 present an algorithm that reduces human
input to a minimum and can create DNA nanostructures with a greater diversity of sizes, edge
lengths and topologies. The fully automated procedure is named DAEDALUS (DNA origami
sequence design algorithm for user-defined structures) after the virtuoso craftsman and the creator
of labyrinths in Greek mythology.
In brief, the procedure starts by representing the target structure as a polyhedral mesh
and computing the corresponding 3D graph (step i) and the ‘spanning tree’ (step ii). The spanning
tree algorithm routes the linear scaffold strand through the target shape with an Eulerian circuit
(step iii) and finally generates staple strand sequences (step iv) for sealing the origami structure
(Fig. 1a). The wireframe motif is based on two interconnected DNA double helices (i.e., double-
crossover (DX) molecules), allowing more structural robustness than the previously reported top-
down objects with single duplex edges7. After designing the shape and obtaining the sequences,
researchers fabricate the structures using standard annealing routines. The DAEDALUS software
(http://daedalus-dna-origami.org/) not only generates a list of staple strands but also produces
atomistic models of the designed structures, whereas the CanDo software (http://cando-dna-
origami.org/)5,6 can be used to simulate shapes of the objects in aqueous solution.
Bathe and colleagues1 demonstrate the power of DAEDALUS by creating numerous
Platonic, Archimedean, Johnson, and Catalan solids as well as objects with higher structural
complexity, such as asymmetric constructs and polyhedra with non-spherical topologies (Fig. 1b).
They also show how to produce linear scaffold strands of the required sequence and length by a
facile asymmetric PCR. The scaffolds they use are shorter or longer than the conventional M13
phage DNA—a circular strand that has a proven track record as a suitable scaffold3 but that is
strictly fixed in size (7,249 nucleotides). Custom-length scaffolds that minimize the excess of
single-stranded DNA may help increase folding yields and structural integrity in low-salt
conditions.
The authors verify that 45 different shapes are correctly folded by atomic force
microscopy and single-particle cryo-electron microscopy, further supported by 3D reconstruction
compared to model predictions. These studies also reveal detailed structural features, such as the
chirality of the vertex twist. The scaffold routing is counter-clockwise around each face due to the
preference of the DNA major groove to point inwards at vertices, and indeed, the cryo-electron
microscopy reconstructions confirm this expected geometry. Interestingly, the objects can be folded
in the presence of low concentrations of cations (magnesium or sodium) and in phosphate-buffered
saline alone. The reported constructs are stable in phosphate-buffered saline and in other cell-
compatible buffers (e.g., Dulbecco’s Modified Eagle Media with fetal bovine serum) for up to 6
hours, an important prerequisite for any biological application9.
Although the above results indicate that relatively stable wireframe DNA objects can
be designed and synthesized, a goal for future work will be to increase the durability of structures
for certain implementations. Along these lines, the authors speculate that further generalizations of
their technique could include edge design with arbitrary cross-sections. This feature would make
structures mechanically sturdier and help in obtaining closed-surface topologies, which are needed,
for example, to encapsulate cargo in DNA containers10. Another intriguing route toward in vivo
applications would be to develop automated methods for genetically encoding DNA nanostructures
in order to synthesize them in living cells.
Overall, the work of Bathe and colleagues1 adds to a growing body of research that is
broadening the structural diversity of DNA nanostructures. Various applications of the technology
are already beginning to emerge. The structural similarities between rigid, cage-like DNA
nanostructures and virus particles or other protein cages suggest approaches in which nanostructures
decorated with appropriate targeting peptides are used to deliver drugs to particular cell types or to
trigger immune responses9,10 (Fig. 1c).
But it is perhaps in the area of materials engineering that the majority of near-term
applications lie. Rigid, closed DNA nanostructures might serve, for instance, as ‘molds’ for the
production of custom-shaped metal nanoparticles11 (Fig. 1c). In fact, because they offer such an
astoundingly high degree of structural order— similar to that found in proteins3—DNA
nanostructures may also find use as scaffolds for precisely positioning other pivotal molecules, such
as catalytic enzymes. As methods such as DAEDALUS move us closer to the in silico design of
ever more complex DNA nanostructures at atomic resolution, the era of fully programmable
nanoscale materials comes increasingly into view4.
Competing financial interests
The authors declare no competing financial interests.
1. Veneziano, R. et al. Designer nanoscale DNA assemblies programmed from the top
down. Science 352, 1534 (2016).
2. Rothemund, P.W.K. Folding DNA to create nanoscale shapes and patterns. Nature
440, 297–302 (2006).
3. Linko, V. & Dietz, H. The enabled state of DNA nanotechnology. Curr. Opin.
Biotechnol. 24, 555–561 (2013).
4. Jones, M.R., Seeman, N.C. & Mirkin, C.A. Programmable materials and the nature of
the DNA bond. Science 347, 1260901 (2015).
5. Castro, C.E. et al. A primer to scaffolded DNA origami. Nat. Methods 8, 211–219
(2011).
6. Pan, K. et al. Lattice-free prediction of three-dimensional structure of programmed
DNA assemblies. Nat. Commun. 5, 5578 (2014).
7. Benson, E. et al. DNA rendering of polyhedral meshes at the nanoscale. Nature 523,
411–444 (2015).
8. Benson, E. et al. Computer-aided production of scaffolded DNA nanostructures from
flat sheet meshes. Angew. Chem. Int. Ed. DOI:10.1002/anie.201602446 (2016).
9. Surana, S., Shenoy, A.R. & Krishnan, Y. Designing DNA nanodevices for
compatibility with the immune system of higher organisms. Nat. Nanotechnol. 10, 741–747 (2015).
10. Linko, V., Ora, A. & Kostiainen, M.A. DNA nanostructures as smart drug-delivery
vehicles and molecular devices. Trends Biotechnol. 33, 586–594 (2015).
11. Sun, W. et al. Casting inorganic structures with DNA molds. Science 346, 1258361
(2014).
Figure 1. Fully automated, top-down design procedure to create arbitrary DNA nanostructures. (a)
Outline of the DAEDALUS1 workflow, which generates DNA nanostructures with rigid double-
crossover molecule edges. (b) Examples of Platonic, Archimedean, Johnson, Catalan and
miscellaneous DNA shapes produced using the method. (c) Potential applications facilitated by the
technique. Figure reproduced with permission from ref. 1, The American Association for the
Advancement of Science.
Functionalize core
Functionalize surface Virus-mimicking targeting
c
Cage-directed synthesis of nanoparticles
Platonic Archimedean Johnson Catalan Miscellaneous
Target polyhedron Schlegel diagram Routing scheme Sequence design Atomic model
(i) Compute graph
(ii) Compute spanning tree
(iii) Route scaffold
(iv) Assign staples
b
a
Veikko Linko and Mauri Kostiainen are at the Biohybrid Materials, Department of Biotechnology
and Chemical Technology, Aalto University, Finland.
e-mail: veikko.linko@aalto.fi; mauri.kostiainen@aalto.fi
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