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Folding DNA to create nanoscale shapes and patterns

16 Mar 2006-Nature (Nature Publishing Group)-Vol. 440, Iss: 7082, pp 297-302
TL;DR: This work describes a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes, which can be programmed to bear complex patterns such as words and images on their surfaces.
Abstract: 'Bottom-up fabrication', which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).

Summary (2 min read)

Design of scaffolded DNA origami

  • The design of a DNA origami is performed in five steps, the first two by hand and the last three aided by computer (details in Supplementary Note S1).
  • Thus for the scaffold to raster progressively from one helix to another and onto a third, the distance between successive scaffold crossovers must be an odd number of half turns.
  • Once the geometric model and a folding path are designed, they are represented as lists of DNA lengths and offsets in units of halfturns.
  • Staples reverse direction at these crossovers; thus crossovers are antiparallel, a stable configuration well characterized in DNA nanostructures 16 .
  • The pattern of merges is not unique; different choices yield different final patterns of nicks and staples.

Folding M13mp18 genomic DNA into shapes

  • To test the method, circular genomic DNA from the virus M13mp18 was chosen as the scaffold.
  • Six different folds were explored; Fig. 2 gives their folding paths and their predicted and experimentally observed DNA structures.
  • By AFM, 13% of structures were well-formed squares (out of S ¼ 45 observed structures) with aspect ratios from 1.00 to 1.07 and bore the expected pattern of crossovers (Fig. 2a , upper AFM image).
  • A range of aspect ratios implied a gap size from 0.9 to 1.2 nm; later designs assume 1 nm.
  • Even when bridging staples at the vertices were not used, a large number of sharp triangles were well-formed (55%, S ¼ 22).

Patterning and combining DNA origami

  • In addition to binding the DNA scaffold and holding it in shape, staple strands provide a means for decorating shapes with arbitrary patterns of binary pixels.
  • Patterns are created by mixing appropriate subsets of these strands.
  • Whether missing pixels represent real defects or artefacts of imaging is unknown; sequential AFM images occasionally showed '1' pixels that later converted irreversibly to '0' pixels, suggesting tip-induced damage.
  • Controlled combination of shapes was achieved by designing 'extended staples' that connected shapes along their edges.

Discussion

  • The scaffolded self-assembly of DNA strands has been used to create linear structures 17, 18 and proposed as a method for creating arbitrary patterns 18, 19 .
  • M13mp18 is essentially a natural sequence that has a predicted secondary structure which is more stable (lower in energy) than similar random sequences (Supplementary Note S8).
  • Further, each correct addition of a staple organizes the scaffold for subsequent binding of adjacent staples and precludes a large set of undesired secondary structures.
  • In addition, each structure required about one week to design and one week to synthesize ; the mixing and annealing of strands required a few hours.
  • These ideas suggest that scaffolded DNA origami could find use in fields as diverse as molecular biology and device physics.

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© 2006 Nature Publishing Group
Folding DNA to create nanoscale shapes
and patterns
Paul W. K. Rothemund
1
‘Bottom-up fabrication’, which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is
widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high
complexity, matching that routinely achieved by ‘top-down’ methods. The self-assembly of DNA molecules provides an
attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into
arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase
single-stranded scaffold and by choosing over 200 short oligonucleotide ‘staple strands’ to hold the scaffold in place.
Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA
structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars
with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be
programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures
can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which
constitutes a 30-megadalton molecular complex).
In 1959, Richard Feynman put forward the challenge of writing the
Encyclopaedia Britannica on the head of a pin
1
, a task which he
calculated would require the use of dots 8 nm in size. Scanning probe
techniques have essentially answered this challenge: atomic force
microscopy
2
(AFM) and scanning tunnelling microscopy
3,4
(STM)
allow us to manipulate individual atoms. But these techniques create
patterns serially (one line or one pixel at a time) and tend to require
ultrahigh vacuum or cryogenic temperatures. As a result, methods
based on self-assembly are considered as promising alternatives that
offer inexpensive, parallel synthesis of nanostructures under mild
conditions
5
. Indeed, the power of these methods has been demon-
strated in systems based on components ranging from porphyrins
6
to
whole viral particles
7
. However, the ability of such systems to yield
structures of high complexity remains to be demonstrated. In
particular, the difficulty of engineering diverse yet specific binding
interactions means that most self-assembled structures contain just a
few unique positions that may be addressed as ‘pixels’.
Nucleic acids can help overcome this probl em: the exqu isite
specificity of Watson–Crick base pairing allows a combinatorially
large set of nucleotide sequences to be used when designing binding
interactions. The field of ‘DNA nanotechnology’
8,9
has exploited this
property to create a number of more complex nanostructures,
including two-dimensional arrays with 8–16 unique positions and
less than 20 nm spacing
10,11
, as well as three-dimensional shapes such
as a cube
12
and truncated octahedron
13
. However, because the
synthesis of such nanostructures involves interactions between a
large number of short oligonucleotides, the y ield of complete
structures is highly sensitive to stoichiometry (the relative ratios of
strands). The synthesis of relatively complex structures was thus
thought to require multiple reaction steps and purifications, with the
ultimate complexity of DNA nanostructures limited by necessarily
low yields. Recently, the controlled folding of a long single DNA
strand into an octahedron was reported
14
, an approach that may be
thought of as ‘single-stranded DNA origami’. The success of this work
suggested that the folding of long strands could, in principle, proceed
without many misfoldings and avoid the problems of stoichiometry
and purification associated with methods that use many short DNA
strands.
I now present a versatile and simple one-pot’ method for using
numerous short single strands of DNA to direct the folding of a long,
single strand of DNA into desired shapes that are roughly 100 nm in
diameter and have a spatial resolution of about 6 nm. I demonstrate
the generality of this method, which I term ‘scaffolded DNA origami’,
by assembling six different shapes, such as squares, triangles and five-
pointed stars. I show that the method not only provides access to
structures that approximate the outline of any desired shape, but also
enables the creation of structures with arbitrarily shaped holes or
surface patterns composed of more than 200 individual pixels. The
patterns on the 100-nm-sized DNA shapes thus have a complexity
that is tenfold higher than that of any previously self-assembled
arbitrary pattern and comparable to that achieved using AFM and
STM surface manipulation
4
.
Design of scaffolded DNA origami
The design of a DNA origami is performed in five steps, the first two
by hand and the last three aided by computer (details in Supplemen-
tary Note S1). The first step is to build a geometric model of a DNA
structure that will approximate the desired shape. Figure 1a shows an
example shape (outlined in red) that is 33 nm wide and 35 nm tall.
The shape is filled from top to bottom by an even number of parallel
double helices, idealized as cylinders. The helices are cut to fit the
shape in sequential pairs and are constrained to be an integer number
of turns in length. To hold the helices together, a periodic array of
crossovers (indicated in Fig. 1a as small blue crosses) is incorporated;
these crossovers designate positions at which strands running along
one helix switch to an adjacent helix and continue there. The
resulting model approximates the shape within one turn (3.6 nm)
in the x-direction and roughly two helical widths (4 nm) in the
ARTICLES
1
Departments of Computer Science and Computation & Neural Systems, California Institute of Technology, Pasadena, California 91125, USA.
Vol 440|16 March 2006|doi:10.1038/nature04586
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© 2006 Nature Publishing Group
y-direction. As noticed before in DNA lattices
15
, parallel helices in
such structures are not close-packed, perhaps owing to electrostatic
repulsion. Thus the exact y-resolution depends on the gap between
helices. The gap, in turn, appears to depend on the spacing of
crossovers. In Fig. 1a crossovers occur every 1.5 turns along alter-
nating sides of a helix, but any odd number of half-turns may be used.
In this study, data are consistent with an inter-helix gap of 1 nm
for 1.5-turn spacing and 1.5 nm for 2.5-turn spacing, yielding a
y-resolution of 6 or 7 nm, respectively.
Conceptually, the second step (illustrated in Fig. 1b) proceeds by
folding a single long scaffold strand (900 nucleotides (nt) in Fig. 1b)
back and forth in a raster fill pattern so that it comprises one of the
two strands in every helix; progression of the scaffold from one helix
to another creates an add itional set of crossovers, the ‘scaffold
crossovers (indicated by small red crosses in Fig. 1b). The funda-
mental constraint on a folding path is that the scaffold can form a
crossover only at those locations where the DNA twist places it at a
tangent point between helices. Thus for the scaffold to raster
progressively from one helix to another and onto a third, the distance
between successive scaffold crossovers must be an odd number of half
turns. Conversely, where the raster reverses direction vertically and
returns to a previously visited helix, the distance between scaffold
crossovers must be an even number of half-turns. Note that the
folding path shown in Fig. 1b is compatible with a circular scaffold
and leaves a ‘seam (a contour which the path does not cross).
Once the geometric model and a folding path are designed, they
are represented as lists of DNA lengths and offsets in units of half-
turns. These lists, along with the DNA sequence of the actual scaffold
to be used, are input to a computer program. Rather than assuming
10.5 base pairs (bp) per turn (which corresponds to standard B-DNA
twist), the program uses an integer number of bases between periodic
crossovers (for example, 16 bp for 1.5 turns). It then performs the
third step, the design of a set of ‘staple strands’ (the coloured DNA
strands in Fig . 1c) that provide Watson–Crick complements for the
Figure 1 | Design of DNA origami. a, A shape (red) approximated by parallel
double helices joined by periodic crossovers (blue). b, A scaffold (black) runs
through every helix and forms more crossovers (red). c, As first designed,
most staples bind two helices and are 16-mers. d, Similar to c with strands
drawn as helices. Red triangles point to scaffold crossovers, black triangles to
periodic crossovers with minor grooves on the top face of the shape, blue
triangles to periodic crossovers with minor grooves on bottom. Cross-
sections of crossovers (1, 2, viewed from left) indicate backbone positions
with coloured lines, and major/minor grooves by large/small ang les between
them. Arrows in c point to nicks sealed to create green strands in d. Yellow
diamonds in c and d indicate a position at which staples may be cut and
resealed to bridge the seam. e, A finished design after merges and
rearrangements along the seam. Most staples are 32-mers spanning three
helices. Insets show a dumbbell hairpin (d) and a 4-T loop (e), modifications
used in Fig. 3.
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© 2006 Nature Publishing Group
scaffold and create the periodic crossovers. Staples reverse direction
at these crossovers; thus crossovers are antiparallel, a stable configu-
ration well characterized in DNA nanostructures
16
. Note that the
crossovers in Fig. 1c are drawn somewhat misleadingly, in that single-
stranded regions appear to span the inter-helix gap even though the
design leaves no bases unpaired. In the assembled structures, helices
are likely to bend gently to meet at crossovers so that only a single
phosphate from each backbone occurs in the gap (as ref. 16 suggests
for similar structures). Such small-angle bending is not expected to
greatly affect the width of DNA origami (see also Supplementary
Note S2).
The minimization and balancing of twist strain between crossovers
is complicated by the non-integer number of base pairs per half-turn
(5.25 in standard B-DNA) and the asy mmetric nature of the helix (it
has major and minor grooves). Therefore, to balance the strain
15
caused by representing 1.5 turns with 16 bp, periodic crossovers are
arranged with a glide symmetry, namely that the minor groove faces
alternating directions in alternating columns of periodic crossovers
(see Fig. 1d, especially cross-sections 1 and 2). Scaffold crossovers are
not balanced in this way. Thus in the fourth step, the twist of scaffold
crossovers is calculated and their position is changed (typically by a
single bp) to minimize str ain; staple sequences are recomputed
accordingly. Along seams and some edges the minor groove angle
(1508) places scaffold crossovers in tension with adjacent periodic
crossovers (Fig . 1d, cross-section 2); such situations are left
unchanged.
Wherever two staples meet there is a nick in the backbone. Nicks
occur on the top and bottom faces of the helices, as depicted in
Fig. 1d. In the final step, to give the staples larger binding domains
with the scaffold (in order to achieve higher binding specificity and
higher binding energy which results in higher melting temperatures),
pairs of adjacent staples are merged across nicks to yield fewer, longer,
staples (Fig. 1e). To strengthen a seam, an additional pattern of
breaks and merges may be imposed to yield staples that cross the
seam; a seam spanned by staples is termed ‘bridged’. The pattern of
merges is not unique; different choices yield different final patterns of
nicks and staples. All merge patterns create the same shape but, as
shown later, the merge pattern dictates the type of grid underlying
any pixel pattern later applied to the shape.
Folding M13mp18 genomic DNA into shapes
To test the method, circular genomic DNA from the virus M13mp18
was chosen as the scaffold. Its naturally single-stranded 7,249-nt
sequence was examined for secondar y structure, and a hairpin with a
20-bp stem was found. Whether staples could bind at this hairpin was
unknown, so a 73-nt region containing it was avoided. When a linear
scaffold was required, M13mp18 was cut (in the 73-nt region) by
digestion with BsrBI restriction enzyme. While 7,176 nt remained
available for folding, most designs did not fold all 7,176 nt; short
(#25 nt) ‘remainder strands’ were added to complement unused
sequence. In general, a 100-fold excess of 200–250 staple and
remainder strands were mixed with scaffold and annealed from
Figure 2 | DNA origami shapes. Top row, folding paths. a, square;
b, rectangle; c, star ; d, disk with three holes; e, triangle with rectangular
domains; f, sharp triangle with trapezoidal domains and bridges between
them (red lines in inset). Dangling curves and loops represent unfolded
sequence. Second row from top, diagrams showing the bend of helices at
crossovers (where helices touch) and away from crossovers (where helices
bend apart). Colour indicates the base-pair index along the folding path; red
is the 1st base, purple the 7,000th. Bottom two rows, AFM images. White
lines and arrows indicate blunt-end stacking. White brackets in a mark the
height of an unstretched square and that of a square stretched vertically (by a
factor .1.5) into an hourglass. White features in f are hairpins; the triangle
is labelled as in Fig. 3k but lies face down. All images and panels without scale
bars are the same size, 165 nm £ 165 nm. Scale bars for lower AFM images:
b,1
m
m; cf, 100 nm.
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© 2006 Nature Publishing Group
95 8Cto208Cin,2 h. When samples were deposited on mica, only
folded DNA structures stuck to the surface while excess staples
remained in solution; AFM imaging thus proceeded under buffer
without prior purification. Six different folds were explored; Fig. 2
gives their folding paths and their predicted and experimentally
observed DNA structures. (Models and staple sequences are given in
Supplementary Note S3, final designs appear in Supplementary Note
S12. Experimental methods are given in Supplementary Note S4,
results described here but not shown are in Supplementary Note S5.)
Of the products imaged by AFM, a particular structure was con-
sidered qualitatively ‘well-formed’ if it had no defect (hole or
indentation in the expected outline) greater than 15 nm in diameter.
For each fold the fraction of well-formed structures, as a percentage
of all distinguishable structures in one or more AFM fields, was
calculated as a rough estimate of yield. I note that while some
structures classified as well-formed had 15-nm defects, most had
no defects greater than 10 nm in diameter.
First, a simple 26-helix square was designed (Fig. 2a). The square
had no vertical reversals in raster direction, required a linear scaffold,
and used 2.5-turn crossover spacing. Most staples were 26-mers that
bound each of two adjacent helices as in Fig. 1c, but via 13 bases
rather than 8. The design was made assuming a 1.5-nm inter-helix
gap; an aspect ratio of 1.05 (93.9 nm £ 89.5 nm) was expected. By
AFM, 13% of structures were well-formed squares (out of S ¼ 45
observed structures) with aspect ratios from 1.00 to 1.07 and bore the
expected pattern of crossovers (Fig. 2a, upper AFM image). Of the
remaining structures, ,25% were rectangular fragments, and ,25%
had an hourglass shape that showed a continuous deformation of the
crossover lattice (Fig. 2a, lower AFM image). Sequential imaging
documented the stretching of a square into an hourglass, suggesting
that hourglasses were originally squares that stretched upon deposition
or interaction with the AFM tip. No subsequent designs exhibited
stretching. Other designs had either a tighter 1.5-turn spacing with
32-mer staples spanning three helical domains (Fig. 2b–d, f) or
smaller domains that appeared to slide rather than stretch (Fig. 2e).
To test the formation of a bridged seam, a rectangle was designed
(Fig. 2b) according to the scheme outlined in Fig. 1e using 1.5-turn
crossover spacing , 32-mer staples and a circular scaffold. As seen in
Fig. 2b, the central seam and associated pattern of crossovers was
easily visualized (upper AFM image). Rectangles stacked along their
vertical edges, often forming chains up to 5
m
m long (lower AFM
image). The yield of well-formed rectangles was high (90%, S ¼ 40),
and so rectangles were used to answer basic questions concerning
inter-helix gaps, base-stacking, defects and stoichiometry. AFM drift
Figure 3 | Patterning and combining DNA origami. a, Model for a pattern
representing DNA, rendered using hairpins on a rectangle (Fig. 2b). b, AFM
image. One pixelated DNA turn (,100 nm) is 30£ the size of an actual DNA
turn (,3.6 nm) and the helix appears continuous when rectangles stack
appropriately. Letters are 30 nm high, only 6£ larger than those written
using STM in ref. 3; 50 billion copies rather than 1 were formed. c, d, Model
and AFM image, respectively, for a hexagonal pattern that highlights the
nearly hexagonal pixel lattice used in ai. ei, Map of the western
hemisphere, scale 1:2 £ 10
14
, on a rectangle of different aspect ratio.
Normally such rectangles aggregate (h) but 4-T loops or tails on edges (white
lines in e) greatly decrease stacking (i). jm, Two labellings of the sharp
triangle show that each edge may be distinguished. In ju, pixels fall on a
rectilinear lattice. nu, Combination of sharp triangles into hexagons
(n, p, q) or lattices (o, ru). Diag rams (n, o) show positions at which staples
are extended (coloured protrusions) to match complementary single-
stranded regions of the scaffold (coloured holes). Models (p, r) permit
comparison with data (q, s). The largest lattice obser ved comprises only
30 triangles (t). u shows close association of triangles (and some breakage).
d and f were stretched and sheared to correct for AFM drift. Scale bars:
h, i,1
m
m; q, su, 100 nm.
ARTICLES NATURE|Vol 440|16 March 2006
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© 2006 Nature Publishing Group
often distorts aspect ratios so that inter-helix gaps cannot be inferred
from the aspect ratio of a single rectangle. A range of aspect ratios
implied a gap size from 0.9 to 1.2 nm; later designs assume 1 nm.
Whatever the exact value, it is consistent: aspect ratios were invariant
along stacked chains with dozens of rectangles. Such stacking was
almost completely abolished by omitting staples along vertical edges.
On the other hand, stacking across the seam of an unbridged
rectangle (as in Fig. 1c) kept 65% of structures (S ¼ 40) well-formed;
the rest showed some degree of dislocation at the seam. Other defects,
such as the intentional omission of single staples, could be visualized
as 5–10-nm holes. However, sharp tips and high tapping amplitudes
were required; rep eated scanning create d holes difficult to dis-
tinguish from holes due to missing strands. This effect also increased
uncertainty when stoichiometry was varied. When staple excesses of
approximately 100:1 and 9:1 were used, the frequencies of 5–10-nm
holes (a few per rectangle) were indistinguishable. At 2:1, rectangles
were similar; perhaps a greater fraction were malformed. At 1.5:1,
rectangles formed but had holes up to ,10% of their area in size. At a
1:1 ratio, ,1% of structures were rectangular.
To demonstrate the creation of arb itrary shapes, a five-pointed star
was designed with 1.5-turn spacing, 32-mer staples and a linear
rather than circular scaffold (Fig. 2c). Designed assuming a 1.5-nm
inter-helix gap (the work was carried out before the gap for 1.5-turn
spacing was measured), the stars are somewhat squat (Fig. 2c, upper
AFM image). Still, the stars show that the width of a shape may be
approximated to within one DNA turn. Many of the structures
observed were star fragments (Fig. 2c, lower AFM image), and only
11% (S ¼ 70) were well-formed. The low yield of stars (and squares,
see above) may be due to strand breakage occurring during BsrBI
digestion or subsequent steps to remove the enzyme; when untreated
circular scaffold was folded into stars, 63% (S ¼ 43) were well-
formed. To show that DNA origami need not be topological disks,
and that scaffolds can be routed arbitrarily through shapes, a three-
hole disk was designed (Fig. 2d). Although the shape approximated is
symmetric, the folding path is highly asymmetric and has five distinct
seams. Unlike the rectangles, which rarely break or fold, three-hole
disks exhibit several characteristic deformations (Fig. 2d, lower AFM
image); still, 70% (S ¼ 90) were well-formed.
DNA origami is not limited to the approximation of shapes by
raster fill: some shapes can be created more exactly by combining
distinct raster fill domains in non-parallel arrangements. Figure 2e
shows a triangle built from three separate, 2.5-turn spacing rec-
tangular domains; only single covalent bonds along the scaffold hold
the domains together. But the desired equiangular triangles (upper
AFM image) were rarely observed (,1%, S ¼ 199). As seen in the
lower AFM image, stacking caused rectangular domains of separate
triangles to bind; this effect and the flexibility of the single-bond
joints at the vertices may account for the ease with which these
triangles deform. To solve these problems, ‘sharp triangles’, built
from trapezoidal domains with 1.5-turn spacing, were designed
(Fig. 2f). The slanted edges of the trapezoids meet at the triangle
vertices and allow the addition of bridg ing staples along these
interfaces. Sharp triangles remained separated and equiangular
(Fig. 2f, lower AFM image); 88% were well-formed (S ¼ 78). Even
when bridging staples at the vertices were not used, a large number of
sharp triang les were well-formed (55%, S ¼ 22). These ‘weakened’
sharp triangles provided the most stringent test of the estimated
inter-helix gap, because too high or low an estimate would have
caused gaps or overlaps between trapezoids. Gaps of 10 nm occasion-
ally appeared but overlaps were never observed, suggesting that 1 nm
may be a slight underestimate of the inter-helix gap.
Patterning and combining DNA origami
In addition to binding the DNA scaffold and holding it in shape,
staple strands provide a means for decorating shapes with arbitrary
patterns of binary pixels. Given a shape, the original set of staples is
taken to represent binary ‘0’s; a new set of labelled staples, one for
each original staple, is used to represent binary ‘1’s. Patterns are
created by mixing appropriate subsets of these strands. In this way,
any desired pattern can be made.
In principle, a variety of DNA modifications
for example, biotin
or fluorophores
could serve as labels. Here, dumbbell hairpins’
(Fig. 1d inset, Supplementary Note S6), designed to avoid dimeriza-
tion at high concentration, were added to the middle of 32-mer
staples at the position of merges made during design. Depending on
the merge pattern, the resulting pixel pattern was either rectilinear,
with adjacent columns of hairpins on alternate faces of the shape, or
staggered and nearly hexagonally packed, with all hairpins on the
same face. In AFM images labelled staples give greater height contrast
(3 nm above the mica) than unlabelled staples (,1.5 nm), which
results in a pattern of light ‘1’ and dark ‘0’ pixels. Several patterns
(Fig. 3), each with ,200 pixels, illustrat e the generality of this
technique.
Yields of patterned origami were similar to those of unpatterned
origami; for the pattern in Fig. 3a, 91% (S ¼ 85) of rectangles were
well-formed. Because rectilinear patterns imaged poorly, only stag-
gered patterns were examined quantitatively. Distances measured
between pairs of ‘1’ pixels in alternating columns (two pixel widths:
11.5 ^ 0.9 nm, mean ^ s.d., n ¼ 26) and adjacent rows (one pixel
height: 6.6 ^ 0.5 nm, n ¼ 24) are consistent with the theoretically
expected pixel size of 5.4 nm £ 6 nm. Most defects take the form of
‘missing pixels’; that is, pixels that should image as ‘1’s but image as
‘0’s instead. 94% of ‘1’ pixels (of 1,080 observed) were visualized.
Whether missing pixels represent real defects or artefacts of imaging
is unknown; sequential AFM images occasionally showed ‘1’ pixels
that later converted irreversibly to ‘0’ pixels, suggesting tip-induced
damage. Stoichiometric errors, synthetic errors, or unwanted sec-
ondary structure are not implicated for any particular strand, as the
position of missing pixels appeared random (Fig. 3b, f and g).
Stacking of shapes along blunt-ended helices provides an uncon-
trolled mechanism for the creation of larger structures (Fig. 3b).
Instead of removing staples on the edge of a rectangle to avoid
stacking (as described previously), 4-T hairpin loops (four thymines
in a row, Fig. 1e, inset) or 4-T tails can be added to edge staples
(Fig. 3e, f); stacked chains of 3–5 rectangles still formed (Fig. 3g),
but 30% of rectangles (S ¼ 319) occurred as monomers (Fig. 3i).
Without hairpins, all rectangles occurred in aggregates (Fig. 3h).
Controlled combination of shapes was achieved by des igning
extended staples’ that connected shapes along their edges. To create
a binding interaction between two particular edges, extended staples
were designed by merging and breaking normal staples along these
edges (Supplementary Note S7). Starting with sharp triangles, this
approach was used to create finite (hexagons; Fig. 3n, p, q) as well as
periodic structures (triangular lattice; Fig. 3o, r–u). I note that the
successful combination of shapes (unlike the successful formation of
individual shapes) is in principle very sensitive to the concentrations
of extended staples (which should ideally be equal to that of the
scaffold). Poor stoichiometry may play a role in the poor yield of
hexagons (,2%, S ¼ 70) and lattices (not measured).
Discussion
The scaffolded self-assembly of DNA strands has been used to create
linear structures
17,18
and proposed as a method for creating arbitrary
patterns
18,19
. But the widespread use of scaffolded self-assembly, and
in particular the use of long DNA scaffolds in combination with
hundreds of short strands, has been inhibited by several misconcep-
tions: it was assumed that (1) sequences must be optimized
20
to avoid
secondary structure or undesired binding interactions, (2) strands
must be highly purified, and (3) strand concentrations must be
precisely equimolar. These three criteria are important for the
formation of many DNA nanostructures and yet all three are ignored
in the present method. For example, M13mp18 is essentially a
natural sequence that has a predicted secondary structure which is
more stable (lower in energy) than similar random sequences
NATURE|Vol 440|16 March 2006 ARTICLES
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Citations
More filters
01 Dec 1991
TL;DR: In this article, self-assembly is defined as the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds.
Abstract: Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds. Molecular self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecular surfaces in biological aggregates is a central concern in structural biochemistry. Self-assembly is also emerging as a new strategy in chemical synthesis, with the potential of generating nonbiological structures with dimensions of 1 to 10(2) nanometers (with molecular weights of 10(4) to 10(10) daltons). Structures in the upper part of this range of sizes are presently inaccessible through chemical synthesis, and the ability to prepare them would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.

2,591 citations

Journal ArticleDOI
21 May 2009-Nature
TL;DR: This work demonstrates the design and assembly of nanostructures approximating six shapes—monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross, and heterotrimeric wireframe icosahedra with precisely controlled dimensions.
Abstract: Molecular self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase 'scaffold strand' that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometre scale.

2,247 citations

Journal ArticleDOI
17 Feb 2012-Science
TL;DR: An autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell surface inputs for conditional, triggered activation, and reconfiguring its structure for payload delivery is described.
Abstract: We describe an autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell surface inputs for conditional, triggered activation, and reconfiguring its structure for payload delivery. The device can be loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. We implemented several different logical AND gates and demonstrate their efficacy in selective regulation of nanorobot function. As a proof of principle, nanorobots loaded with combinations of antibody fragments were used in two different types of cell-signaling stimulation in tissue culture. Our prototype could inspire new designs with different selectivities and biologically active payloads for cell-targeting tasks.

1,865 citations

Journal ArticleDOI
15 Mar 2012-Nature
TL;DR: It is shown that DNA origami enables the high-yield production of plasmonic structures that contain nanoparticles arranged in nanometre-scale helices, and it is found that the structures in solution exhibit defined circular dichroism and optical rotatory dispersion effects at visible wavelengths that originate from the collective plAsmon–plasmon interactions of the nanoparticles positioned with an accuracy better than two nanometres.
Abstract: Matter structured on a length scale comparable to or smaller than the wavelength of light can exhibit unusual optical properties. Particularly promising components for such materials are metal nanostructures, where structural alterations provide a straightforward means of tailoring their surface plasmon resonances and hence their interaction with light. But the top-down fabrication of plasmonic materials with controlled optical responses in the visible spectral range remains challenging, because lithographic methods are limited in resolution and in their ability to generate genuinely three-dimensional architectures. Molecular self-assembly provides an alternative bottom-up fabrication route not restricted by these limitations, and DNA- and peptide-directed assembly have proved to be viable methods for the controlled arrangement of metal nanoparticles in complex and also chiral geometries. Here we show that DNA origami enables the high-yield production of plasmonic structures that contain nanoparticles arranged in nanometre-scale helices. We find, in agreement with theoretical predictions, that the structures in solution exhibit defined circular dichroism and optical rotatory dispersion effects at visible wavelengths that originate from the collective plasmon-plasmon interactions of the nanoparticles positioned with an accuracy better than two nanometres. Circular dichroism effects in the visible part of the spectrum have been achieved by exploiting the chiral morphology of organic molecules and the plasmonic properties of nanoparticles, or even without precise control over the spatial configuration of the nanoparticles. In contrast, the optical response of our nanoparticle assemblies is rationally designed and tunable in handedness, colour and intensity-in accordance with our theoretical model.

1,838 citations

Journal ArticleDOI
TL;DR: Here, this work reviews DNA strand-displacement-based devices, and looks at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.
Abstract: The programmable and reliable hybridization of DNA strands has enabled the preparation of a wide variety of structures. This Review discusses how, in addition to these static assemblies, the process of displacing — and ultimately replacing — strands also makes possible the construction of dynamic systems such as logic gates or autonomous walkers.

1,520 citations


Cites background from "Folding DNA to create nanoscale sha..."

  • ...Since then, DNA origami technology [44] has enabled the construction of significantly longer tracks with more complex geometry [50, 96] leading to correspondingly longer processive walks and integration of multiple different kinds of DNA nanomotors....

    [...]

  • ...Several recent advances in structural DNA selfassembly have been based on the DNA origami technology [44], which uses short oligonucleotide “staple” strands to fold a long single-stranded “scaffold” (typically the m13 viral genome) into two- and threedimensional shape of interest [9]....

    [...]

References
More filters
Journal ArticleDOI
01 Jan 1985-Gene
TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.

14,954 citations

Journal ArticleDOI
TL;DR: The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large by making use of universally available web GUIs (Graphical User Interfaces).
Abstract: The abbreviated name,‘mfold web server’,describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces),the server circumvents the problem of portability of this software. Detailed output,in the form of structure plots with or without reliability information,single strand frequency plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information,but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/ mfold. This URL will be referred to as ‘MFOLDROOT’.

12,535 citations

Journal ArticleDOI
29 Nov 1991-Science
TL;DR: The ability to prepare structures in the upper part of this range of sizes would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.
Abstract: Molecular self-assembly is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates joined by noncovalent bonds. Molecular self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures. Understanding self-assembly and the associated noncovalent interactions that connect complementary interacting molecular surfaces in biological aggregates is a central concern in structural biochemistry. Self-assembly is also emerging as a new strategy in chemical synthesis, with the potential of generating nonbiological structures with dimensions of 1 to 10(2) nanometers (with molecular weights of 10(4) to 10(10) daltons). Structures in the upper part of this range of sizes are presently inaccessible through chemical synthesis, and the ability to prepare them would open a route to structures comparable in size (and perhaps complementary in function) to those that can be prepared by microlithography and other techniques of microfabrication.

3,119 citations

Journal ArticleDOI
D. M. Eigler1, E. K. Schweizer1
01 Apr 1990-Nature
TL;DR: In this paper, Binnig and Rohrer used the scanning tunnelling microscope (STM) to position individual xenon atoms on a single-crystal nickel surface with atomic pre-cision.
Abstract: SINCE its invention in the early 1980s by Binnig and Rohrer1,2, the scanning tunnelling microscope (STM) has provided images of surfaces and adsorbed atoms and molecules with unprecedented resolution The STM has also been used to modify surfaces, for example by locally pinning molecules to a surface3 and by transfer of an atom from the STM tip to the surface4 Here we report the use of the STM at low temperatures (4 K) to position individual xenon atoms on a single-crystal nickel surface with atomic pre-cision This capacity has allowed us to fabricate rudimentary structures of our own design, atom by atom The processes we describe are in principle applicable to molecules also In view of the device-like characteristics reported for single atoms on surfaces5,6, the possibilities for perhaps the ultimate in device miniaturization are evident

2,765 citations


"Folding DNA to create nanoscale sha..." refers background in this paper

  • ...These are (1) strand invasion, (2) an excess of staples, (3) cooperative effects and (4) design that intentionally does not rely on binding between staples....

    [...]

  • ...But the widespread use of scaffolded self-assembly, and in particular the use of long DNA scaffolds in combination with hundreds of short strands, has been inhibited by several misconceptions: it was assumed that (1) sequencesmust be optimized(20) to avoid secondary structure or undesired binding interactions, (2) strands must be highly purified, and (3) strand concentrations must be precisely equimolar....

    [...]

Journal ArticleDOI
06 Aug 1998-Nature
TL;DR: The design and observation of two-dimensional crystalline forms of DNA that self-assemble from synthetic DNA double-crossover molecules that create specific periodic patterns on the nanometre scale are reported.
Abstract: Molecular self-assembly presents a `bottom-up' approach to the fabrication of objects specified with nanometre precision. DNA molecular structures and intermolecular interactions are particularly amenable to the design and synthesis of complex molecular objects. We report the design and observation of two-dimensional crystalline forms of DNA that self-assemble from synthetic DNA double-crossover molecules. Intermolecular interactions between the structural units are programmed by the design of `sticky ends' that associate according to Watson-Crick complementarity, enabling us to create specific periodic patterns on the nanometre scale. The patterned crystals have been visualized by atomic force microscopy.

2,713 citations