, 1177 (2012);
et al.Yonggang Ke
Three-Dimensional Structures Self-Assembled from DNA Bricks
This copy is for your personal, non-commercial use only.
clicking here.colleagues, clients, or customers by
, you can order high-quality copies for your
If you wish to distribute this article to others
The following resources related to this article are available online at
here.following the guidelines
can be obtained by
Permission to republish or repurpose articles or portions of articles
Updated information and services,
): December 13, 2012 www.sciencemag.org (this information is current as of
version of this article at:
including high-resolution figures, can be found in the online
can be found at:
Supporting Online Material
related to this article
A list of selected additional articles on the Science Web sites
, 13 of which can be accessed free:
cites 43 articles
1 articles hosted by HighWire Press; see:
This article has been
This article appears in the following
registered trademark of AAAS.
is aScience2012 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience
on December 13, 2012
Self-Assembled from DNA Bricks
Yonggang Ke,1,2,3Luvena L. Ong,1,4William M. Shih,1,2,3Peng Yin1,5*
We describe a simple and robust method to construct complex three-dimensional (3D) structures by
using short synthetic DNA strands that we call “DNA bricks.” In one-step annealing reactions, bricks
with hundreds of distinct sequences self-assemble into prescribed 3D shapes. Each 32-nucleotide
brick is a modular component; it binds to four local neighbors and can be removed or added
independently. Each 8–base pair interaction between bricks defines a voxel with dimensions of
2.5 by 2.5 by 2.7 nanometers, and a master brick collection defines a “molecular canvas” with
dimensions of 10 by 10 by 10 voxels. By selecting subsets of bricks from this canvas, we
constructed a panel of 102 distinct shapes exhibiting sophisticated surface features, as well as
intricate interior cavities and tunnels.
lecular structures and devices (1–31). Structures
have been designed by encoding sequence com-
plementarity in DNA strands in such a manner
that by pairing up complementary segments,
the strands self-organize into a prescribed tar-
get structure under appropriate physical condi-
tions (1). From this basic principle, researchers
have created diverse synthetic nucleic acid struc-
tures (27–30) such as lattices (4, 6, 8–10, 25),
ribbons (15), tubes (6, 15, 25, 26), finite two-
dimensional (2D) and 3D objects with defined
shapes (2, 9–11, 13, 16–19, 22, 23, 26), and
macroscopic crystals (20). In addition to static
structures, various dynamic systems have been
constructed (31), including switches (5), walkers
(7, 14, 21), circuits (12, 14, 24), and triggered as-
sembly systems (14). Additionally, because DNA
and RNA can be interfaced with other functional
molecules in a technologically relevant fashion,
synthetic nucleic acid structures promise diverse
applications; researchers are using nucleic acid
structures and devices to direct spatial arrange-
ment of functional molecules (6, 25, 32–34),
facilitate protein structure determination (35),
develop bioimaging probes (33, 34), study single-
molecule biophysics (36), and modulate bio-
synthetic and cell-signaling pathways (25, 37).
An effective method for assembling megadalton
nanoscale 2D (11) and 3D shapes (16–19, 23) is
elf-assembly of nucleic acids (DNA and
RNA) provides a powerful approach for
constructing sophisticated synthetic mo-
DNA origami (29), in which a long “scaffold”
strand (often a viral genomic DNA) is folded to
a predesigned shape via interactions with hun-
dreds of short “staple” strands. However, each
distinct shape typically requires a new scaffold
routing design and the synthesis of a different
set of staple strands. In contrast, construction from
standardized small components (such as DNA
tiles) that each can be included, excluded, or re-
placed without altering the rest of the structure—
modular assembly—offers a simpler approach
to constructing shapes. In addition, if all compo-
nents are short strands that can be chemically
synthesized, the resulting structures would have
greater chemical diversity than DNA origami,
which typically contains half biological material
(the scaffold) in mass and half synthetic material
(the staples). Avariety of structures have been as-
sembled by using DNA (3, 4, 6, 8, 10, 13, 15, 20)
and RNA (9, 22, 25) tiles, including periodic
(4, 6, 25) and algorithmic (8) 2D lattices, extended
ribbons (15) and tubes (6, 15, 25), 3D crystals (20),
polyhedra (13, 22), and finite 2D shapes (9, 10).
However, modular self-assembly of finite-sized,
discrete DNA structures has generally lacked the
complexity that DNA origami can offer.
Only recently have researchers demonstrated
finite complex 2D shapes (26) self-assembled
from hundreds of distinct single-stranded tiles
(SSTs) (15). Unlike a traditional multistranded tile
(3, 4, 6, 8–10, 13, 20, 25), which is a well-folded,
compact structure displaying several sticky ends,
an SST is a floppy single-strand DNA composed
entirely of concatenated sticky ends. In one-pot
reactions, hundreds of SSTs self-assemble into
desired target structures mediated by inter-tile
binding interactions; no scaffold strand is re-
quired. The simplicity and modularity of this
approach allowed the authors to build more than
100 distinct shapes by selecting subsets of tiles
from a common 2D “molecular canvas.” This
latest success has challenged previous thinking
that modular components, such as DNA tiles,
are not suitable for assembling complex, singu-
larly addressable shapes (38). This presumption
was largely based on a supposed technically
challenging requirement for perfect strand stoi-
chiometry (the relative ratio of the strands). De-
viations from equality were expected to result
in predominating partial structure formation (38).
The surprising success of SST assembly may
have bypassed this challenge via putative slow
and sparse nucleation followed by fast growth
(26), so that a large number of particles com-
plete their formation well before depletion of the
component strand pool.
Here, we generalize the concept of single-
stranded “tiles” to “bricks” and thus extend our
modular-assembly method from 2D to 3D. A
canonical DNA brick is a 32-nucleotide (nt) sin-
gle strand with four 8-nt binding domains (sticky
ends).In simple one-stepannealingreactions, pre-
ly from hundreds of unpurified brick strands that
are mixed together with no tight control of stoi-
chiometry. The modularity of our method en-
abled the construction of 102 distinct structures
bysimplyselectingsubsetsofbricksfrom a com-
mon 3D cuboid molecular canvas consisting of
1000 voxels (fig. S1) (39); each voxel fits 8 base
by 2.7 nm. These structures include solid shapes,
with sophisticated geometries and surface pat-
structures with alternative packing geometries or
using noncanonical brick motifs, demonstrating
lishes DNA bricks as a simple, robust, modular,
3D nanostructures by using only short synthetic
DNA strands. More generally, it demonstrates
how complex 3D molecular structures can be as-
sembled from small, modular components medi-
ated strictly by local binding interactions.
Design of DNA-Brick Structures
and a 3D Molecular Canvas
In our design, a DNA brick is a 32-nt strand that
we conceptualize as four consecutive 8-nt do-
mains (Fig. 1A). Each DNA brick bears a dis-
tinct nucleotide sequence. All DNA bricks adopt
an identical shape when incorporated into the tar-
get structure: two 16-nt antiparallel helices joined
by a single phosphate linkage. The two domains
adjacent to the linkage are designated as “head”
domains, and the other two are designated as “tail”
domains. A DNA brick with a tail domain bear-
ing sequence “a” can interact productively with a
neighboring brick with a complementary “a*” head
domain in a stereospecific fashion. Each pairing
between bricks defines three parallel helices packed
to produce a 90° dihedral angle (Fig. 1B, top); this
angle derives from the approximate 3/4 right-
handed helical twist of 8 bp of DNA.
We introduce a LEGO-like model to depict the
design in a simple manner (Fig. 1B, bottom). The
model intentionally overlooks the detailed helical
1Wyss Institute for Biologically Inspired Engineering, Harvard
University, Boston, MA 02115, USA.2Department of Cancer
Biology, Dana-Farber Cancer Institute, Harvard Medical School,
Harvard University, Boston, MA 02115, USA.3Department of
Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Harvard University, Boston, MA 02115, USA.
4Harvard–Massachusetts Institute of Technology (MIT) Divi-
sion of Health Sciences and Technology, MIT, Cambridge, MA
02139, USA.5Department of Systems Biology, Harvard Med-
ical School, Harvard University, Boston, MA 02115, USA.
*To whom correspondence should be addressed. E-mail:
VOL 33830 NOVEMBER 2012
on December 13, 2012
structure and strand polarity but preserves the as-
pect ratios and some of the orientational con-
straints on interactions between DNA bricks: The
two protruding round plugs, pointing in the same
direction as the helical axes, represent the two tail
domains; the two connected cubes with recessed
must adopt one of two classes of orientation, hori-
to form a 90° angle via hybridization, represented
onlyallowedbetweenaplug anda hole thatcarry
complementary sequences with matching polar-
ity (which is not graphically depicted in the cur-
explicitly tracks the polarity of the DNA bricks
and their stereospecific interaction pattern.
Structural periodicities of the design are il-
lustrated in a 6H (helix) by 6H (helix) by 48B
(bp) cuboid structure (Fig. 1, C and D). Bricks
can be grouped into 8-bp layers that contain their
head domains. Bricks follow a 90° counterclock-
wise rotation along successive 8-bp layers, re-
sulting in a repeating unit with consistent brick
orientation and arrangement every four layers.
For example, the first and fifth 8-bp layers in
Fig. 1D share the same arrangement of bricks.
Within an 8-bp layer, all bricks share the same
orientation and form a staggered arrangement
to tile the layer. On the boundary of each layer,
some DNA bricks are bisected to half-bricks,
representing a single helix with two domains.
The cuboid is self-assembled from DNA bricks
in a one-step reaction. Each brick carries a par-
ticular sequence that directs it to fit only to its
predesigned position. Because of its modular
architecture, a predesigned DNA brick structure
can be used for construction of smaller custom
shapes assembled from subsets of DNA bricks
(Fig. 1E). Detailed strand diagrams for the DNA
brick structures are provided in figs. S3 and S4.
3D molecular canvas. The LEGO-like model
can be further abstracted to a 3D model that con-
tains only positional information of each 8-bp
duplex. A 10H by 10H by 80B cuboid is concep-
tualized as a 3D molecular canvas that contains
10 by 10 by 10 voxels. Each voxel fits an 8-bp
duplex and measures 2.5 by 2.5 by 2.7 nm (Fig.
1F). Based on the 3D canvas, a computer program
first generates a full set of DNA bricks, including
full-bricks and half-bricks that can be used to build
a prescribed custom shape. Using 3D modeling
software, a designer then needs only to define the
target shapes by removing unwanted voxels from
the 3D canvas—a process resembling 3D sculpt-
ing. Subsequently, the computer program analyzes
the shape and automatically selects the correct
subset of bricks for self-assembly of the shape.
Self-Assembly of DNA-Brick Cuboid Structures
Using the above design strategy, we constructed
a wide range of DNA brick structures (39). We
Domain 2Domain 1
= 8 bp
= 8 bp
Subset 1Subset 2
Fig. 1. DesignofDNAbrickstructuresanalogoustostructuresbuiltofLEGO®
bricks. (A) A 32-nt four-domain single-stranded DNA brick. Each domain is
8 nt in length. The connected domains 2 and 3 are “head” domains; domains
1 and 4 are “tail” domains. (B) Each two-brick assembly forms a 90° dihedral
angle via hybridization of two complementary 8-nt domains “a” and “a*”. (C)
A molecular model that shows the helical structure of a 6H by 6H by 48B
cuboid 3D DNA structure. Each strand has a particular sequence, as indicated
by a distinct color. The inset shows a pair of bricks. (D) A LEGO-like model of
the 6H by 6H by 48B cuboid. Each brick has a particular sequence. The color
use is consistent with (B). Half bricks are present on the boundary of each
layer. (E) The 6H by 6H by 48B cuboid is self-assembled from DNA bricks. The
bricks are not interchangeable during self-assembly because of the distinct
sequence of each brick. Using the 6H by 6H by48Bas a3D molecular canvas,
asmallershapecanbedesigned byusing asubsetofthebricks.(F)3Dshapes
by 2.5 nm by 2.7 nm).
30 NOVEMBER 2012VOL 338
on December 13, 2012
first constructed 3D cuboid structures of a variety
of sizes and aspect ratios (Fig. 2).
Random sequence design. The sequences of
DNA bricks were designed by random assign-
ments of base pairs (A-T, C-G) to 3D structures.
We first tested two versions of a 6H by 6H by 64B
cuboid, with either random sequences or special-
ly designed sequences (designed by smoothing
binding energy, minimizing undesired second-
ary structure, and reducing sequence symmetry)
and observed comparable self-assembly yields
(fig. S5). We also tested three sets of random se-
quences using a 4H by 12H by 120B cuboid and
again observed similar assembly yields (figs. S6
and S7; more discussion on domain similarity
of random sequence design is provided in fig.
S8). Thus, random sequences were applied to all
Protector bricks. Including unpaired single
strands at the ends of DNA duplexes has proven
to be effective for mitigating unwanted aggre-
gation that results from blunt-end stacking (11).
An 8-nt single-stranded domain protruded out
from every 5′ or 3′ end of all DNA duplexes in
our 3D structure designs (Fig. 1C). The sequences
of these 8-nt domains were replaced with eight
continuous thymidines to further prevent unde-
sired nonspecific binding interactions between
exposed single-stranded domains. DNA bricks
with modified head or tail poly-T domains are
named “head protectors” or “tail protectors,”
Boundary bricks. A 16-nt half brick could be
merged with a preceding 32-nt full brick along
the direction of its helix to form a 48-nt strand
(figs. S9 to S11). We observed a 1.4-fold improve-
ment in assembly yield for a 6H by 6H by 64B
cuboid when this 48-nt boundary-strand design
was implemented, possibly reflecting accelerated
nucleation of target structure formation. Hence,
this merge strategy was applied to all of our 3D
Assembly and characterization of 6H by 10H
by 128B cuboid. For a detailed characteri-
zation study, we constructed a 6H by 10H by
128B cuboid (Fig. 2A). It consists of 459 strands
(7680 bp, with a molecular weight comparable
with that of an M13-based DNA origami; design
details are provided in figs. S12 and S13). Un-
purified DNA strands were mixed together at
nominally equal ratios without careful adjust-
ment of stoichiometry (39). To determine the
optimal assembly conditions, we tested two an-
nealing ramps (24-hour annealing and 72-hour
annealing), two strand concentrations (100 and
200 nM per strand), and eight MgCl2concen-
trations (10, 20, 30, 40, 50, 60, 70, and 80 mM).
Equal amounts of each sample (2 pmol per strand)
were then subjected to EtBr-stained 2% agarose
gel electrophoresis (fig. S14). The best gel yield
(~4% as calculated by yield = measured mass
of product/mass of all strands) was achieved at
the following conditions: 200 nM per strand,
above gel yield reflects only an approximate esti-
mate for the incorporation ratio of the monomer
For comparison, 4 to 14% gel yield was re-
ported for 3D DNA origami with similar size and
aspect ratios [such as the 10H by 6H by 98B and
other origami cuboids in (40)]. The origami gel
yield was estimated as yield = (scaffold strands
incorporated into product/total scaffold strands);
the loss of excessive staple strands (normally 5-
to 10-fold more than the scaffold strand) was not
taken into account. For DNA bricks, the optimal
40 mM MgCl2was higher than the optimal
MgCl2concentration for 3D origami folding,
which typically is below 30 mM (18). Column-
purified DNA bricks product (~50% recovery
efficiency) (Fig. 2B) migrated as a single band
on agarose gel and appeared under transmis-
sion electron microscopy (TEM) with expected
morphology (Fig. 2C) and measured dimen-
sions of 0.34 nm (T 0.01 nm SD) per base pair
and 2.5 nm (T 0.2 nm SD) per helix width. For
the gel-purified product, “the percentage of in-
tact structures” was estimated at 55% by counting
the ratio of intact particles over all the parti-
cles in TEM images (fig. S16). This percent-
age of intact structures is comparable with the
previously reported percentages of 3D square-
lattice DNA origami (27% for a 6H by 12H by
80B cuboid, 59% for an 8H by 8H by 96B cu-
Special designs can be applied to increase the
assembly yield of the 6H by 10H by 128B cuboid.
“Head protectors” and “tail protectors” appeared
especially unstable because half of their 8-nt do-
mains are unpaired. By merging “head protec-
tors” of the 6H by 10H by 128B cuboid with their
neighboring strands (figs. S17 and S18), a mod-
ified version 6H by 10H by 128B-M cuboid was
obtained and showed 190% improvement in gel
assembly yield and 17% improvement in the
percentage of intact structures under TEM over
the standard 6H by 10H by 128B cuboid (fig.
S19). Thus, 3D structures can be further stabi-
lized by using special design rules, such as this
merging strategy. However, this modification
requires deletions of crossovers between helices,
which may potentially create global or local de-
formations, and was not used for constructions
in the remainder of the paper.
Structures of different sizes. Eighteen distinct
cuboid structures that contain 9, 16, 36, 60, 96,
and 144 helices were designed, annealed using
the optimal conditions previously identified for
the 6H by 10H by 128B cuboid self-assembly, and
characterized through gel and TEM (Fig. 2D and
M 1 2
3H × 3H8H × 12H12H × 12H
123456789 10 11 12
6H × 6H
6H × 10H
4H × 24H
4H × 4H
Fig. 2. Cuboid structures self-assembled from DNA bricks. (A) DNA bricks self-
assembled into a 6H by 10H by 128B cuboid in a one-step thermal annealing
process. (B) Agarose gel electrophoresis showing 50% purification recovery
efficiency of the 6H by 10H by 128B cuboid. Lane M contains the 1-kb ladder.
Lanes 1 and 2 contain unpurified and purified 6H by 10H by 128B cuboid
structures, respectively. The red arrow points to the cuboid product band. (C)
TEM images of gel-purified 6H by 10H by 128B cuboid. Zoomed-in images
(bottom) and corresponding computer-generated graphics (middle) show three
different projection views. (D) Designs and TEM images of 18 cuboids of a
variety of dimensions. Horizontal axis is labeled with the cross-section dimen-
sions of the cuboids; vertical axis is labeled with the lengths of the constituent
helices. The lengths are 48B (shape 18), 64B (shapes 1, 6, 10, 13, and 15),
120B (shapes 16 and 17), 128B (shapes 2, 7, 11, and 14), 256B (shapes 3, 8,
and 12), 512B (shapes 4 and 9), and 1024B (shape 5). Each 3D cylinder
model is drawn proportionally to the relative dimensions of the cuboid; cor-
responding TEM images are shown to the right or above each model.
VOL 33830 NOVEMBER 2012
on December 13, 2012
fig. S20). Additional TEM images are shown
in figs. S21 to S27. Measured dimensions of
intact particles for each structure agree with the
designs (fig. S28). Gel yields varied from <1 to
~80% (figs. S20C and S28). For structures with
the same number of helices, smaller cuboids
exhibited higher assembly yields. The highest
yield (80%) was observed for the smallest ob-
ject, the 3H by 3H by 64B cuboid; the lowest
yields (<1%) were observed for the 8H by 12H
by 120B, 4H by 24H by 120B, and 12H by 12H
by 48B cuboids. The biggest DNA objects con-
structed in this paper are an 8H by 12H by 120B
cuboid (formed by 728 strands) and a 4H by 24H
by 120B cuboid (formed by 710 strands), which
are identical in molecular weight (24,576 nt,
8 MD, and 60% more massive than an M13-
based DNA origami). Increasing the concentra-
tion for the brick strands helped to increase the
yield for a small cuboid, 4H by 4H by 128B
(fig. S29). In some cases, higher molecular weight
bands can be detected above the product band;
these bands are likely multimers caused by non-
specific interactions between assembled products.
For example, for the 6H by 10H by 64B struc-
ture, TEM revealed that an upper band con-
tained dimers of the cuboids (fig. S30). Cuboids
with 32-bp (32B) helices were also tested but
failed to assemble (fig. S20). This is likely due
to the fact that these cuboids contained only
one crossover between each pair of neighboring
helices and hence were less stable.
Complex Shapes Made from a 10 by 10
by 10–Voxel 3D Canvas
Using the 10 by 10 by 10–voxel 3D canvas (Figs.
1F and 3A and fig. S31), we next constructed
102 distinct shapes (Fig. 3), demonstrating the
modularity of the DNA brick strategy.
Shape 1: voxel(1,1,1), voxel(2,1,3), ...
Shape 2: voxel(4,5,4), voxel(10,1,5), ...
Shape 3: voxel(1,1,1), voxel(9,10,8), ...
Shape 1: strand 1, strand 2, strand 3, ...
Shape 2: strand 3, strand 4, strand 5, ...
Shape 3: strand 1, strand 5, strand 6, ...
10 × 10 × 10 canvas
= 8 bp
13 14 15 16
1920 2122 2324 25
43 4445 46 4748 52 53
83 848586 87 8889909192
9596 97 9899
5758 5960 616256
Fig. 3. Shapes made from a 3D molecular canvas. (A) A 10 by 10 by 10–voxel
3Dcanvas.z axisisthe helicalaxis.Eachvoxel(8 bp)measures2.5 by2.5by
2.7 nm. (B) Shapes are designed by editing voxels by using 3D modeling
software. (C) A computer program recognizes the voxel composition of each
shape and generates a list of strands to form this shape. The list then is used
to direct an automated liquid-handling robot to mix the strands. (D) After
annealing, the shapes are characterized by means of agarose gel electro-
indicated by the red arrow. (E) Computer-generated models and TEM images of
generated projection view, an image averaged from six different particles
visualized by using TEM,and arepresentative raw TEM image.More raw images
are shown in figs. S38 to S54. In a number of cases, multiple projections are
presented. Some shapes with cavities or tunnels are depicted with additional
transparent 3D views that highlight the deleted voxels (colored dark gray). For
example, the top right model of shape 32 shows the enclosed cuboid cavity.
30 NOVEMBER 2012 VOL 338
on December 13, 2012
DNA bricks and derivatives. Any brick in the
3D canvas can become either a boundary half
brick (exposed at the edges of a layer and bi-
time, in a custom shape design. Thus, modified
polythymidine-sequence-substitution (change to
sibilities (two types of strands with low occurring
strands (with a total of 138,240 nt) were generated
collection without synthesizing new strands.
Automated design process. By rendering the
3D canvas using 3D modeling software, we can
edit voxels and visualize a shape using a graph-
ical user interface (Fig. 3B). Then, the voxel
information of multiple shapes is interpreted by a
custom program to generate a list of strands in-
volved in the formation of each shape. This list
is subsequently processed to direct an automated
liquid-handling robot to select DNA strands from
source plates and pipette them to the wells of a
product plate, mixing strands for many shapes
in a high-throughput manner (Fig. 3C). The strands
will be subsequently annealed in separate test
tubes to produce the desired structures (Fig. 3D).
The complete design workflow is shown in figs.
S33 and S34. To use existing computational tools
previously developed by other researchers, we
can also convert shapes to caDNAno files (40).
Each shape’s conformation then can be simulated
using CanDo (42), a software tool for comput-
ing 3D structures of DNA origami (fig. S35).
Using the 3D canvas and following the auto-
mated design process, we successfully constructed
102 distinct shapes (gels in figs. S36 and S37;
TEM images of shapes 1 to 100 in Fig. 3E; and
raw TEM images for all the shapes in figs. S38
Shapes 1 to 17. The basic design constraints
were studied by using a group of shapes contain-
ing two 4H by 10H by 80B blocks connected
by a middle “connecting block” (shapes 2 to 17).
The connecting blocks were two-voxel wide along
x axis and systematically designed to possess de-
creasing numbers of voxels along y axis (shapes
2 to 9) or z axis (shapes 10 to 17). Eliminating
voxels along the x axis should have the same
effect as eliminating voxels along the y axis be-
cause of the shape symmetry. Agarose gel elec-
trophoresis revealed that in both systems, as the
connector became overly thin, the gel yields
for the intact structures decreased, and partial
structures (putative unconnected 4H by 10H by
80B blocks) became more prominent (for ex-
ample, in lanes for shapes 8, 9, and 15 to 17 in
fig. S36). However, reducing the number of
voxels along the z axis appeared to decrease the
yield more significantly than along the y axis.
Shape 9, which contained only a 2-voxel con-
nection along the y axis, gave 6% gel yield. In
contrast, the yield for shape 17 (2-voxel along
the z axis) dropped to 1%. Overall, these obser-
vations suggest safe design criteria of at least
two continuous voxels along the x axis or y axis
(2 helices) and three z axis voxels (24 bp) for
stable features. However, as demonstrated in fol-
lowing experiments, smaller features (for exam-
ple, two z axis voxels, shapes 33 to 37; one x axis
or y axis voxel, shapes 64 to 74) can still stably
exist in certain shapes in which these features are
presumably reinforced by other voxels in close
were designed including z direction extrusions
ofsimple geometricshapes(shapes18to 23) and
more intricate objects (shapes 24 to 31; also, shape
102 in fig. S54). Gel yields and TEM images of
these objects provided more knowledge of the
design space of our methodology. For example,
shapes 26 and 27, which both contained 3-helix-
thick appendages anchored only on one edge,
wereoccasionallyfoundwithout these protrusions
or with them but containing defects. Thus, such
appeared to be less stable than were the better-
supported or thicker features.
Closed-cavity shapes 32 to 42. Previously, a
few examples of 3D DNA origami with closed
cavities were demonstrated, including a box (16),
a tetrahedron (17), a sphere, and an ellipsoid (23).
We created a series of “empty boxes” with differ-
ent sizes of cuboid cavities (shapes 32 to 37) as
well as more intricate cavity shapes (such as a
square ring, cross, and triangle; shapes 38 to 42).
Open-cavity shapes 43 to 62. We constructed
shapes with a single open cavity (tunnel) of vary-
ing width, depth, and geometry (shapes 43 to 53)
and multiple-parallel cavities (shapes 54 to 56).
Shapes with noncrossing perpendicular tunnels
(shape 57), turning and branching tunnels (shape
58), and crossing tunnels (shapes 59, 60; also,
shape 101 in fig. S54) were also demonstrated.
Furthermore, we constructed tunnel-containing cu-
varying external views from different angles, as
demonstrated by shapes 60 to 62.
Features-on-solid-base shapes 63 to 100. So-
phisticated features were designed on a solid
base, including a full set of 10 Arabic numerals
(shapes 65 to 74) and 26 lowercase letters for
the English alphabet (shapes 75 to 100). Two
concentric ring structures (shapes 63 and 64)
and the numerals (shapes 65 to 74) contained
features as thin as one voxel (2.5 nm), suggest-
ing that the design criteria (for example, thin
structures tend to fail) are contingent on the
surrounding environment of a particular feature.
These shapes also highlight the capacity of creat-
ing extruded features that would otherwise be
unattainable via 2D assembly (26).
For most shapes, assembly yields were be-
tween a few percent and 30% [figs. S36 and S37;
in comparison, yields of five 3D DNA origami
structures were reported as 7 to 44% (18)]. Only
five shapes had assembly yields higher than 30%;
three shapes had assembly yields lower than 1%.
In spite of our success in making a variety
of intricate 3D shapes, some shapes exhibited
undesired properties. For example, shapes 60 to
62 only showed <1% of intact particles in TEM
images; some fine features of a shape (such as
the two wings of shape 27) could be damaged
or even completely missing if the shape was ex-
tracted from an agarose gel band. We also ob-
served four failed designs that did not produce
clear product bands on agarose gels (fig. S55A).
Two features-on-solid-base designs showed strong
bands on agarose gels (fig. S55B), and were of
the expected size in TEM images. However, their
features were not clearly resolved under TEM,
suggesting that the shapes may have formed, but
the features were too subtle to be visualized.
Generality of DNA Brick Self-Assembly
To explore the generality of the DNA brick
assembly framework, we constructed struc-
tures with brick motifs other than the 32-nt ca-
nonical brick motif. These structures include
those with alternative lattice geometries that have
been previously demonstrated by DNA origami
(11, 18, 43).
Single-layer (2D) structures. Conceptually, a
single-layer structure can be constructed by “ex-
traction” of a layer from a 3D brick structure
[Fig. 4A and fig. S56, comparison with a 2D
single-stranded tile rectangle design (26)]. A
30H by 1H by 126B rectangle was intention-
ally modified to be 10.5 bp per turn instead of
10.67 bp per turn (for 3D design) in order to
get a relatively flat structure (fig. S57). Gel yield
was estimated to be 18% (fig. S58), which is com-
parable with 2D single-stranded tile structures
(26). TEM (Fig. 4B) and atomic force micros-
copy (AFM) (Fig. 4C) revealed expected rectan-
gle structures. On the basis of AFM images, the
dimensions were measured as 0.31 nm (T 0.01 nm
SD) per base pair and 2.6 nm (T 0.3 nm SD) per
3D honeycomb-lattice structures. We then
created 10.8-bp per turn (33.3° twist per base
pair) honeycomb-lattice (HC) and hexagonal-
lattice (HL) DNA structures. Four types of four-
domain DNA strands were designed for HC
structures (Fig. 4, D and E). A 6H by 6H by 84B-
HC structure was successfully constructed and
characterized (Fig. 4F and fig. S59). Particles
in TEM images were measured to be 13 nm
(T 0.9 nm SD) by 22 nm (T 1.0 nm SD) by 29 nm
(T 1.2 nm SD). Assembly yield was estimated to
be 30% (fig. S60).
3D hexagonal-lattice DNA structures. Two
types of strands are used to build a HL struc-
ture: a linear strand with multiple 9-nt domains
and an 18-nt strand with two 9-nt domains that
are connected by a crossover (Fig. 4, G and H).
VOL 338 30 NOVEMBER 2012
on December 13, 2012
A 6H by 7H by 108B-HL structure was con-
structed and characterized (Fig. 4I and fig. S61).
Particles in TEM images were measured to be
13 nm (T 0.8 nm SD) by 18 nm (T 1.1 nm SD) by
35 nm (T 2.2 nm SD). Assembly yield was es-
timated to be 26% (fig. S62).
Other brick motifs. We also constructed a
6H by 10H by 64B cuboid that arranges brick
strands in an alternating fashion between layers
(figs. S63 and S64) and two 6H by 6H by 64B
cuboids that implement two other brick motif
designs (figs. S65 and S66). One design is based
on “chopping” the scaffold of a DNA origami
to short strands (fig. S65A). The other adopts
standardized motifs that are each 32 nt long and
have two crossovers (fig. S65B). These designs
further demonstrate the versatility of DNA brick
DNA bricks provide a simple, modular, and ro-
bust framework for assembling complex struc-
tures from short strands. Simplicity: A canonical
brick is a standardized 32-nt single strand com-
posed of four 8-nt binding domains; bricks in-
teract via simple local binding rules. Modularity:
With no scaffold present, an assembly of bricks
has a modular architecture; each brick can be
added or removed independently. Robustness:
The assembly process is robust to variations in
sequence composition (random sequences are
used), strand synthesis (unpurified strands suf-
fice), and stoichiometry (no tight control is re-
quired). Together, the simple and standardized
motif, modular architecture, and robust perform-
ance permit straightforward automation of the
design and construction process. A software tool
takes as input a 3D shape specification and di-
rects a liquid-handling robot to select and mix
presynthesized brick strands to form the shape.
Using a 1000-voxel canvas, 102 diverse shapes
were rapidly prototyped. These shapes demon-
strate a new level of geometrical sophistication,
as exemplified by the intricate tunnel and cavity
The DNA brick framework is not restricted
to the canonical 32-nt motif and can be gen-
eralized to include various other motifs (Fig.
4), enabling the construction of 3D structure
with diverse lattice-packing geometries. In addi-
tion, previously demonstrated single-stranded tiles
(15, 26) can be viewed as a special case of bricks
in which each pair of neighboring bricks form a
180° angle. For comparison, in hexagonal-, square-,
and honeycomb-lattice structures, neighboring
bricks form 60°, 90°, and 120° angles, respec-
tively. These different angles are achieved by
changing the domain lengths of bricks. Further-
more, neighboring bricks may be merged into a
longer strand, which may facilitate nucleation or
strengthen structurally weak positions. The DNA
brick (and single-stranded tile) method differs from
previous multistranded tiles in that each brick
monomer is a floppy single strand and only
folds into a bricklike shape when incorporated
into the assembly. It also differs from DNA ori-
gami by not using a scaffold strand. However,
DNA origami can also be related to the brick
framework, in which half of the bricks are con-
catenated into a long scaffold (fig. S65A). The
successes of constructions that use only short
strands (as in bricks) and those that include a
long scaffold (as in origami) together suggest a
full spectrum of motif possibilities with strands
of diverse lengths: Longer strands may provide
better structural support, and shorter ones may
provide finer modularity and features; the eclec-
tic use of both may lead to the most rapid pro-
gression toward greater complexity.
The DNA brick structures constructed here
are still far below the size limit allowed by se-
quence uniqueness. Making the conservative
assumption (by neglecting the contribution of
cooperativity) that every domain must display a
different sequence, a structure using canonical
32-nt, four-domain bricks could potentially reach
a size of 8 nt by 48(524,288 nucleotides). In our
experiments, the assembly process appeared to
tolerate (sparse) identical domains (fig. S8), fur-
ther expanding the potential obtainable size. Fur-
ther exponential increase in size could potentially
be achieved by using bricks with longer domains
or by encoding algorithmic growth patterns (8) in
6H × 7H × 108B-HL
30H × 1H × 126B
6H × 6H × 84B-HC
Top layer of 6H × 6H × 48B
Fig. 4. Generality of DNA brick self-assembly. (A to C) The design and
construction of a single-layer brick structure. (A) DNA bricks of the top layer
of the 6H by 6H by 48B cuboid in Fig. 1D, with the crossovers to the layer
below removed. (B) TEM images of a 30H by 1H by 126B rectangle. Top
right inset shows the model of the design. Bottom right inset contains a
zoomed-in image of the structure. (C) AFM images of the 30H by 1H by
126B rectangle. Inset contains a zoomed-in image of the structure. (D to I)
The designs and constructions of 3D honeycomb-lattice [(D) to (F)] and
hexagonal-lattice [(G) to (I)] brick structures. [(D) and (G)] The strands used
for (D) honeycomb-lattice and (G) hexagonal-lattice self-assembly. The num-
ber of nucleotides in each domain is indicated in the left panel. [(E) and (H)]
Strand diagrams of (E) an 84-bp honeycomb-lattice structure and (H) a 54-bp
hexagonal-lattice structure. The right bottom image depicts an enlarged image
of the circled helix bundle. Strand colors match those described on the right
side of (D) or (G). Numbers indicate DNA helices. [(F) and (I)] TEM images of
(F) a 6H by 6H by 84B-HC hexagonal-lattice structure and (I) a 6H by 7H by
108B-HL 3D hexagonal-lattice structure. 3D model and zoomed-in images of
different projection views are shown to left.
30 NOVEMBER 2012VOL 338
on December 13, 2012
the assembly. However, in practice, low yields Download full-text
were already observed for larger designs (up to
24,576 nucleotides attempted thus far). Solving
this challenge may require improvements in struc-
ture and sequence design, enzymatic synthesis for
higher-quality strands, optimized thermal or iso-
thermal (44) annealing conditions, and a detailed
understanding and perhaps explicit engineering
of the kinetic assembly pathways (8, 14, 44) of
DNA brick structures.
The DNA brick structure, with its modular ar-
chitecture, sophisticated geometry control, and
synthetic nature, will further expand the range of
applications and challenges that nucleic acid nano-
technology has already started to address—for
example, to arrange technologically relevant guest
molecules into functional devices (6, 25, 32–34),
to serve as programmable molecular probes and
instruments for biological studies (33, 34, 36),
to render spatial control for biosynthesis of use-
ful products (25), to function as smart drug deliv-
ery particles (37), and to enable high-throughput
nanofabrication of complex inorganic materials
for electronics or photonics applications (6, 32).
The modularity of the brick structure may facil-
itate rapid prototyping of diverse functional nano-
devices. Its sophisticated and refined geometrical
control may enable applications that require high-
precision arrangements of guest molecules. Be-
cause the brick structure is composed entirely of
short synthetic strands (no biologically derived
scaffold), it is conceivable to make bricks by using
synthetic informational polymers other than the
natural form of DNA. Such polymers may in-
clude L-DNA (26), DNAwith chemically modi-
fied backbones or artificial bases, or chemically
synthesized or in vitro (or even in vivo) transcribed
RNA. This material diversity may potentially
produce nanostructures with not only prescribed
shapes but also designer chemical (or bio-
chemical) properties (such as nuclease resistance
or reduced immunogenicity) that would be useful
for diverse applications requiring the structure to
function robustly in complex environments,
such as in living cells or organisms.
References and Notes
1. N. C. Seeman, J. Theor. Biol. 99, 237 (1982).
2. J. H. Chen, N. C. Seeman, Nature 350, 631 (1991).
3. T. J. Fu, N. C. Seeman, Biochemistry 32, 3211 (1993).
4. E. Winfree, F. Liu, L. A. Wenzler, N. C. Seeman, Nature
394, 539 (1998).
5. B. Yurke, A. J. Turberfield, A. P. Mills Jr., F. C. Simmel,
J. L. Neumann, Nature 406, 605 (2000).
6. H. Yan, S. H. Park, G. Finkelstein, J. H. Reif, T. H. LaBean,
Science 301, 1882 (2003).
7. W. B. Sherman, N. C. Seeman, Nano Lett. 4, 1203
8. P. W. K. Rothemund, N. Papadakis, E. Winfree, PLoS Biol.
2, e424 (2004).
9. A. Chworos et al., Science 306, 2068 (2004).
10. S. H. Park et al., Angew. Chem. Int. Ed. 45, 735
11. P. W. K. Rothemund, Nature 440, 297 (2006).
12. G. Seelig, D. Soloveichik, D. Y. Zhang, E. Winfree, Science
314, 1585 (2006).
13. Y. He et al., Nature 452, 198 (2008).
14. P. Yin, H. M. T. Choi, C. R. Calvert, N. A. Pierce, Nature
451, 318 (2008).
15. P. Yin et al., Science 321, 824 (2008).
16. E. S. Andersen et al., Nature 459, 73 (2009).
17. Y. Ke et al., Nano Lett. 9, 2445 (2009).
18. S. M. Douglas et al., Nature 459, 414 (2009).
19. H. Dietz, S. M. Douglas, W. M. Shih, Science 325, 725
20. J. Zheng et al., Nature 461, 74 (2009).
21. T. Omabegho, R. Sha, N. C. Seeman, Science 324, 67
22. I. Severcan et al., Nat. Chem. 2, 772 (2010).
23. D. Han et al., Science 332, 342 (2011).
24. L. Qian, E. Winfree, Science 332, 1196 (2011).
25. C. J. Delebecque, A. B. Lindner, P. A. Silver, F. A. Aldaye,
Science 333, 470 (2011).
26. B. Wei, M. Dai, P. Yin, Nature 485, 623 (2012).
27. C. Lin, Y. Liu, S. Rinker, H. Yan, ChemPhysChem 7, 1641
28. N. B. Leontis, A. Lescoute, E. Westhof, Curr. Opin.
Struct. Biol. 16, 279 (2006).
29. W. M. Shih, C. Lin, Curr. Opin. Struct. Biol. 20, 276
30. N. C. Seeman, Annu. Rev. Biochem. 79, 65 (2010).
31. D. Y. Zhang, G. Seelig, Nat. Chem. 3, 103 (2011).
32. A. Kuzyk et al., Nature 483, 311 (2012).
33. H. M. T. Choi et al., Nat. Biotechnol. 28, 1208 (2010).
34. C. Lin et al., Nat. Chem. 4, 832 (2012).
35. M. J. Berardi, W. M. Shih, S. C. Harrison, J. J. Chou,
Nature 476, 109 (2011).
36. N. D. Derr et al., Science 338, 662 (2012).
37. S. M. Douglas, I. Bachelet, G. M. Church, Science 335,
38. P. W. K. Rothemund, E. S. Andersen, Nature 485, 584
39. Materials and methods, supplementary figures and texts,
and DNA sequences are available as supplementary
materials on Science Online.
40. S. M. Douglas et al., Nucleic Acids Res. 37, 5001
41. Y. Ke et al., J. Am. Chem. Soc. 131, 15903 (2009).
42. C. E. Castro et al., Nat. Methods 8, 221 (2011).
43. Y. Ke, N. V. Voigt, K. V. Gothelf, W. M. Shih, J. Am.
Chem. Soc. 134, 1770 (2012).
44. R. Schulman, B. Yurke, E. Winfree, Proc. Natl. Acad.
Sci. U.S.A. 109, 6405 (2012).
Acknowledgments: The authors thank M. Dai for technical
assistance; E. Winfree, B. Wei, and S. Woo for discussions; and
D. Pastuszak for assistance in draft preparation. This work is
supported by an Office of Naval Research (ONR) Young
Investigator Program award N000141110914, an ONR
grant N000141010827, an Army Research Office grant
W911NF1210238, an NSF CAREER award CCF1054898, an
NIH Director’s New Innovator award 1DP2OD007292, and
a Wyss Institute Faculty Startup Fund to P.Y., and by a Wyss
Institute Faculty Grant, ONR grants N000014091118 and
N000141010241, and an NIH Director’s New Innovator
award 1DP2OD004641 to W.M.S.. L.L.O. is supported by an
NSF graduate research fellowship. Y.K. conceived the
project, designed and performed the experiments, analyzed
the data, and wrote the paper; L.L.O. designed and performed
the experiments, analyzed the data, and wrote the paper;
W.M.S. conceived the project, discussed the results, and
wrote the paper; P.Y. conceived, designed, and supervised
the study, interpreted the data, and wrote the paper. The
DNA sequences for the nanostructures can be found in the
supplementary materials. A provisional patent has been
filed based on this work.
Materials and Methods
Figs. S1 to S66
Tables S1 to S20
11 July 2012; accepted 16 October 2012
A Reconciled Estimate of Ice-Sheet
Andrew Shepherd,1* Erik R. Ivins,2* Geruo A,3Valentina R. Barletta,4Mike J. Bentley,5
Srinivas Bettadpur,6Kate H. Briggs,1David H. Bromwich,7René Forsberg,4Natalia Galin,8
Martin Horwath,9Stan Jacobs,10Ian Joughin,11Matt A. King,12,27Jan T. M. Lenaerts,13Jilu Li,14
Stefan R. M. Ligtenberg,13Adrian Luckman,15Scott B. Luthcke,16Malcolm McMillan,1
Rakia Meister,8Glenn Milne,17Jeremie Mouginot,18Alan Muir,8Julien P. Nicolas,7John Paden,14
Antony J. Payne,19Hamish Pritchard,20Eric Rignot,18,2Helmut Rott,21Louise Sandberg Sørensen,4
Ted A. Scambos,22Bernd Scheuchl,18Ernst J. O. Schrama,23Ben Smith,11Aud V. Sundal,1
Jan H. van Angelen,13Willem J. van de Berg,13Michiel R. van den Broeke,13David G. Vaughan,20
Isabella Velicogna,18,2John Wahr,3Pippa L. Whitehouse,5Duncan J. Wingham,8Donghui Yi,24
Duncan Young,25H. Jay Zwally26
We combined an ensemble of satellite altimetry, interferometry, and gravimetry data sets using
common geographical regions, time intervals, and models of surface mass balance and
glacial isostatic adjustment to estimate the mass balance of Earth’s polar ice sheets. We find that
there is good agreement between different satellite methods—especially in Greenland and
West Antarctica—and that combining satellite data sets leads to greater certainty. Between 1992
and 2011, the ice sheets of Greenland, East Antarctica, West Antarctica, and the Antarctic
Peninsula changed in mass by –142 T 49, +14 T 43, –65 T 26, and –20 T 14 gigatonnes year−1,
respectively. Since 1992, the polar ice sheets have contributed, on average, 0.59 T 0.20 millimeter
year−1to the rate of global sea-level rise.
els (1, 2) and oceanic conditions. They occur as
luctuations in the mass of the polar ice
sheets are of considerable societal impor-
tance, because they affect global sea lev-
a consequence of their internal dynamics and
changes in atmospheric and oceanic conditions
(3–5). Analysis of the geological record sug-
gests that past climatic changes have precipitated
VOL 33830 NOVEMBER 2012
on December 13, 2012