6792 Chem. Commun., 2010, 46, 6792–6794 This journal is c The Royal Society of Chemistry 2010

Exterior modification of a DNA tetrahedronw

Chuan Zhang,aMin Su,

bYu He,

aYujun Leng,

bAlexander E. Ribbe,

aGuansong Wang,

c

Wen Jiangband Chengde Mao*

a

Received 5th July 2010, Accepted 5th August 2010

DOI: 10.1039/c0cc02363a

This paper reports an introduction of extra structural features

into self-assembled DNA polyhedra.

DNA, as a molecular self-assembly system, has been programmed

to assemble into a range of well-defined nanostructures.1–4 In

the last several years, a wave of effort has been devoted to

developing strategies for assembly of three-dimensional (3D)

DNA nanostructures.5–17 Among them, a biomimetic approach

has been developed to assemble symmetric DNA polyhedra. As

in the self-assembly of viral capsids, this strategy relies on self-

limiting associations of finite numbers of identical components

(symmetric DNA star motifs or tiles). We have applied it to

assemble DNA tetrahedra, hexahedra (cubes), dodecahedra,

icosahedra, and buckyballs.17–20 However, the resulting nano-

structures lack appendage groups that can serve as docking sites

for guest objects such as proteins and nanoparticles. In contrast,

the component proteins of viral capsids contain surface groups

for biological functions. Many viral particles exhibit spikes that

stretch out from viral capsids and play important roles, for

example, specifically attaching to host cell surfaces. Can we

assemble DNA polyhedra with similar spikes? In other words,

can we decorate DNA polyhedra with spikes? Those spikes

should not be involved in the polyhedra assembly, but provide

sites for introducing additional functions. A simple form of

spikes can be a short stem–loop structure (hairpin). Such

structures could potentially perform some interesting roles,

such as introducing DNAzymes21 or aptamers22 to catalyze

chemical reactions or bind specific ligands. To demonstrate the

feasibility of such decoration, we have introduced short hairpins

onto the exterior surface of a DNA tetrahedron, the simplest

DNA polyhedron.

The exterior modification is realized by inserting an extra

segment (encoding a hairpin structure, or spike) into one

component DNA strand (Fig. 1). In our previous study, we

used three unique DNA strands (L: blue–red; M: green; and

S: black) to assemble the tetrahedral structure. Upon cooling

from 95 to 25 1C, the individual single strands associate into

3-point-star tiles. The DNA tile possesses a 3-fold rotational

(C3) symmetry. The tile contains seven DNA strands but only

three different sequences due to the C3 symmetry. Its three

component branches are identical to one another and each has

a pair of complementary sticky-ends. Through sticky-

end association, the tiles assemble into tetrahedra. In the

current work, the M strand has been modified to contain a

16-base-long insert (colored purple), which will fold into a

hairpin structure under native conditions. The hairpin consists

of a 5-basepair-long stem and a 4-T single-stranded loop. To

prevent the short duplex stem from stacking onto other DNA

duplexes, two extra bases (Ts) are introduced at the 50

of the hairpin. In the 3-point-star tile, the hairpin is located

a half turn away from both the crossover point and the tile

center.

Balancing the flexibility and rigidity of a DNA motif is

critically important for DNA self-assembly.23 Compared with

a regular 3-point-star motif, the hairpins introduce additional

flexibilities to the tiles because of the hairpin-containing

3-branched structures. To compensate the extra flexibility,

the length of the central single-stranded loop (colored red) is

reduced from the original 5 bases to 4 bases (Fig. S1). Longer

than 4 bases, the motif is too flexible and only dimers of the

tiles form. Shorter than 4 bases, the motifs become too stiff

Fig. 1 Self-assembly of a DNA tetrahedron with hairpin spikes.

DNA single strands (L, M, and S) stepwisely assemble into symmetric

3-point-star motifs with hairpins (tiles) and then into a hairpin-

tetrahedron in a one-pot process. Note that there are three single-

stranded loops (colored red) in the center of the complex to introduce

flexibility to the hairpin-modified 3-point-star motifs. The purple

segment in the second strand will form short hairpin structures during

self-assembly. All hairpins stretch out from the struts of the DNA

tetrahedron (purple) near the vertices with a three-fold rotational

symmetry.

aDepartment of Chemistry, Purdue University, West Lafayette,Indiana 47907, USA. E-mail: mao@purdue.edu;Fax: +1-765-494-0239; Tel: +1-765-494-4098

bMarkey Center for Structural Biology and Department of BiologicalSciences, Purdue University, West Lafayette, Indiana 47907, USA

c Institute of Respiratory Diseases, Xinqiao Hospital,The Third Military Medical University, Chongqing, Chinaw Electronic supplementary information (ESI) available: DNAsequences, experimental procedures. See DOI: 10.1039/c0cc02363a

COMMUNICATION www.rsc.org/chemcomm | ChemComm

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This journal is c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 6792–6794 6793

and assemble into large aggregates that either have very slow

mobility during polyacrylamide gel electrophoresis (PAGE) or

can not penetrate into the gel matrix at all.

DNA self-assembly was performed according to previously

reported protocols.17 Briefly, the component DNA strands

were mixed at a ratio of 1 : 3 : 3 (L :M : S) in a Tris–acetic

acid–EDTA–Mg2+ buffer and the mixture solution was slowly

cooled from 95 to 25 1C over 24 h. The assembled DNA

structures were characterized by non-denaturing PAGE. As

shown in the gel, all component DNA strands associate

together into a single complex. The complex has a slightly

lower electrophoretic mobility than the original, bare tetra-

hedron (Fig. S1). It is consistent with the fact that the spiked

tetrahedron has a higher molecular weight (by 192 bases) than

the bare tetrahedron. Following PAGE, we have characterized

the spiked DNA tetrahedron by dynamic light scattering

(DLS) and atomic force microscopy (AFM). DLS directly

measures the physical sizes of the DNA complexes in solution.

The apparent radius obtained from DLS is B12.75 � 1.85 nm

(Fig. S2a), slightly larger than the calculated radius of the

bare tetrahedron model (10.90 nm) and the experimentally

measured radius of the bare tetrahedron (10.30 nm), assuming

0.33 nm/base pair for the helical pitch and 2 nm for the

diameter of a DNA duplex, respectively. The observed radius

increase is expected because of the out-pointing spikes

(hairpins). AFM imaging confirms that the DNA complexes

are uniform in size (Fig. S2b). The DNA complexes have a

lateral dimension of B20–23 nm and a height of B2.5 nm.

Particularly, triangular shapes of the particles are fairly

reasonable for a dehydrated and collapsed DNA tetrahedron.

All the above data suggest that the spiked DNA tetrahedron

has formed and the assembly yield is high (480% as estimated

from the PAGE shown in Fig. S1).

To clearly show the spikes on the tetrahedron, we used

cryoEM imaging in conjunction with a single-particle 3D

reconstruction technique to reveal the intact structure of the

DNA complex (Fig. 2). In raw cryoEM micrographs, particles

with the expected size (B23 nm) are clearly visible. Applying

the single particle 3D reconstruction technique to the observed

particles has achieved a tetrahedral map at 26 A resolution. At

such a moderate resolution, a number of expected features are

visible though hairpins can not be clearly resolved. (1)

Each vertex of the hairpin-tetrahedron contains three bumps,

corresponding to the out-pointing hairpins. The hairpins are

1.6 nm long (5 basepairs); it is expected they will appear as

bumps (measured as 1.1 nm high) at the current resolution.

The bumps superficially resemble the spikes on the surfaces of

many spherical viral capsids. (2) The bumps are exhibited on

the tetrahedron surface and are located on the right side

of the strut (containing two pseudo-duplexes) near the

vertices, indicating that all 3-point-star tiles bend into the

same direction during the self-assembling. It is consistent with

our previous observation of the star motifs.20 (3) The class

average images of the particles show a dramatic difference

between the spiked tetrahedron and the bare tetrahedon

(Fig. S4). The hairpins bring extra density to the tetrahedron

particles and result in three bright spots at each vertex in

the class average images and their corresponding computer

generated projections.

In summary, we have introduced hairpins onto the DNA

tetrahedron. The out-pointing hairpins mimic the spikes on

viral capsids. It is the first step towards functionalization of

DNA polyhedra for future applications. 3D nanostructures

are interesting in bionanotechnology because many biological

interactions are strongly related to specific and spatial arrange-

ment and orientation. Hence, the functionalization of the DNA

polyhedra is an important step to investigate such interactions.

The current success opens the door to further modifying the

DNA polyhedra for organizing other nano-objects, such as

proteins or nanoparticles, which are currently under investi-

gation in our group.

This work was supported by the Office of Naval Research

(Award No. N000140910181 and N000140911118) and

the National Science Foundation (0923637). DLS and

AFM studies were carried out in the Purdue Laboratory for

Chemical Nanotechnology (PLCN). The cryo-EM images

were taken in the Purdue Biological Electron Microscopy

Facility and the Purdue Rosen Center for Advanced Com-

puting (RCAC) provided the computational resource for the

3D reconstructions.

Fig. 2 Visualization of the DNA tetrahedron with hairpin spikes

by cryogenic transmission electron microscopy (cryoEM). (a) A

representative image. White boxes indicate the DNA particles.

(b) Raw images of individual particles (bottom) and the corresponding

computer-generated model projections (top). (c) Three views of the

spiked DNA tetrahedron structure reconstructed from cryoEM

images.

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6794 Chem. Commun., 2010, 46, 6792–6794 This journal is c The Royal Society of Chemistry 2010

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