pubs.acs.org/NanoLett

Robust and Photocontrollable DNA Capsules
Using Azobenzenes
Fumiaki Tanaka,*,† Toshio Mochizuki,‡ Xingguo Liang,‡ Hiroyuki Asanuma,‡
Shukichi Tanaka,§ Katsuyuki Suzuki,| Shin-ichi Kitamura,| Akio Nishikawa,⊥
Kumiko Ui-Tei,# and Masami Hagiya††

Graduate School of Information Science and Technology, University of Tokyo, Tokyo 113-8656, Japan, ‡ Graduate
School of Engineering, Nagoya University, Nagoya 464-8601, Japan, § Kobe Advanced-ICT Research Center (KARC),
National Institute of Information and Communications Technology (NICT), Kobe, Hyogo 651-2492, Japan, | JEOL Co. Ltd.,Tokyo 196-8558, Japan, ⊥ Hosei University, and # Graduate School of Science, University of Tokyo,
Tokyo 113-8656, Japan
ABSTRACT Various three-dimensional structures have been created on a nanometer scale using the self-assembly of DNA molecules.
However, ordinary DNA structures breakdown readily because of their flexibility. In addition, it is difficult to control them by inputs
from environments. Here, we construct robust and photocontrollable DNA capsules using azobenzenes. This provides a method toconstruct DNA structures that can survive higher temperatures and can be controlled with ultraviolet irradiation.
KEYWORDS 3D DNA structures, photo control, azobenzene, fast-scanning atomic force microscopy, melting temperatureD

NA strands hybridize with each other in a sequencespecific manner; consequently, the binding between rationally designed DNA strands can becontrolled to allow the assembly of various structures.1 This
makes DNA molecules some of the most powerful building
blocks available for assembling new structures on a nanometer scale such as two-dimensional periodic patterns,2-6
defined complex patterns,7 and tube-shaped structures.8
Many three-dimensional structures have also been createdby folding single-stranded DNA,9 assembling some DNA
sequences,10,11 or using DNA origami12-15 or multibranched
DNA motifs.16-18 These structures are small and complex
enough for applications such as drug delivery and nanoscale
fabrication. However, ordinary DNA structures breakdown
readily because of their flexibility. In addition, it is difficult
to control them by inputs from environments.
Here, we construct robust and photocontrollable DNA
capsules using azobenzenes. The robustness and photocontrollability of the capsules are clearly demonstrated bymeasuring the melting temperature (TM) and using agarose
gel electrophoresis and atomic force microscopy (AFM). Our
DNA capsules with azobenzenes can survive higher temperature. A moving image obtained using fast-scanning AFM
shows how the DNA capsules break into small structures on
ultraviolet (UV) irradiation. Our strategy of using azobenzenes is universal in the sense that other structures canachieve this robustness and photocontrollability by inserting
azobenzenes into their sequences. This enhances the po-tential for DNA structures to be used in technical applications
such as drug-delivery systems as carriers.
To develop DNA capsules, we focused on polyhedra
derived from three-branched structures called three-pointstar motifs, which hybridize to one another and form
tetrahedra, dodecahedra, and buckyballs, depending on
their loop length and concentration.16 Azobenzenes change
their conformation from the trans to the cis form on UV
irradiation at 300-400 nm, and from the cis to the transform on visible light irradiation, at wavelengths >400 nm.
When azobenzenes are inserted into a DNA strand, the cisform prevents the formation of a double-stranded structure.19 Consequently, the hybridization of DNA strands canbe controlled by UV irradiation, allowing the formation ofphotocontrollable DNA structures.20 In our system, azobenzenes were inserted into the sticky ends of three-point-star
motifs. We expected DNA capsules to form and then breakdown into three-point-star motifs on UV irradiation (Figure1). Additionally, azobenzenes can stabilize double-stranded
DNA via stacking interactions.21 This can be used to produce
stable DNA structures.
To confirm the formation of DNA capsules, we observed
the assembled structures of L&M&S-1azo (azobenzene-poor)and L&M-2azo&S-2azo (azobenzene-rich) using AFM (Figure2). Both L&M&S-1azo and L&M-2azo&S-2azo formed sphereshaped DNA capsules in air. As expected, the heights of bothsphere-shaped structures were roughly twice those of threepoint-star motifs (right in Figure 2). However, the yield of
L&M&S-1azo was poorer than that of L&M-2azo&S-2azo.
This was probably due to the position of the azobenzenes
rather than the number of azobenzenes (see Figure 4).

* To whom correspondence should be addressed, fumi95@is.s.u-tokyo.ac.jp.
Received for review: 05/24/2010
Published on Web: 07/29/2010© 2010 American Chemical Society

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zenes in the sequences. This is probably because the positioning of the azobenzenes had a greater effect on theconformation of the assembled structure in L&M&S-1azo
than in L&M-1azo&S.
The photocontrollability of the DNA capsules was clearly
demonstrated using fast-scanning AFM and agarose gelelectrophoresis at 4 °C (Figure 3 and Figure 4). In Figure 3,
the assembled L&M-2azo&S-2azo still formed sphere-shaped
structures for 49 s without UV irradiation (see Supporting
Information for the moving image). This shows that the fastscanning AFM observation did not destroy the DNA capsules.
However, soon after UV irradiation (t ) 50 s), the capsules
started to breakdown into three-point-star motifs, revealing
the photocontrollability of the capsules. The height of a DNA
capsule before UV irradiation was roughly twice that after
UV irradiation (see the lower images in Figure 3). This also
proves that before UV irradiation the assembled structures
successfully formed the expected DNA capsules rather thansingle-layered sheetlike structures.
Figure 4 shows that other DNA capsules can be also
controlled by using UV light. Before UV irradiation, each lane
had two bands: the upper band corresponded to DNA
capsules and the lower band corresponded to thrree-pointstar motifs. The brightness of the bands containing L&M2azo&S-2azo was weak because azobenzenes quench fluorescence. The 12 azobenzenes in one three-point-star motif
formed by L&M-2azo&S-2azo greatly weakened the strengthof the fluorescence from SYBR Gold. As expected, only DNA
capsules formed by azobenzene-containing sequences weredestroyed by UV light irradiation. Despite the stability of
DNA capsules formed by L&M-2azo&S-2azo (see Table 1),
they were more sensitive to UV irradiation than other DNA
capsules. In fact, they were destroyed by UV irradiation for
only 3 s. This is probably because L&M-2azo&S-2azo contained the most azobenzenes (12 in each three-point-star
motif) of the five structures. Thus, azobenzene-rich sequences can both stabilize the assembled structures andincrease their sensitivity to UV light.
In contrast, azobenzene-poor sequences were insensitive
to UV irradiation. Nonetheless, the DNA capsules formed by
L&M&S-1azo could be broken easily. This is probably due
to their instability and the inefficiency of capsule formation.
The structures formed by L&M-1azo&S were difficult tobreak because of their stability (Table 1) and insensitivity to
UV light, because they had the fewest azobenzenes (threein each three-point-star motif).
The yields of DNA capsules were 49.4 ( 3.3% (L&M&S),
45.4 ( 2.0% (L&M-1azo&S), 22.0 ( 5.5% (L&M&S-1azo),39.7 ( 3.5% (L&M-1azo&S-1azo), and 45.9 ( 3.6% (L&M2azo&S-2azo). These were lower than those obtained by He
et al.16 In particular, without azobenzenes (i.e., original DNA
buckyballs), our yield was also worse (49.4%) than theirs(68.9%). We believe that our experimental conditions,especially the DNA concentrations, were not as optimal as
theirs. Additionally, the yields differed depending on the

FIGURE 1. Design of DNA capsules using azobenzenes. The purple
circles and X denote azobenzenes. A 1:3:3 mixture of L, M, and S(by concentration) formed three-point-star motifs, which further
hybridized to one another to form polyhedra.16 The sequences M,
M-1azo, and M-2azo contained 0, 1, and 2 azobenzenes, respectively,
as did the sequences S, S-1azo, and S-2azo. L&r&β (r is M, M-1azo,or M-2azo; β is S, S-1azo, or S-2azo) denotes a mixture of L, r, andβ at a concentration ratio of 1:3:3.

The robustness of the DNA capsules made with azobenzenes was confirmed by measuring TM. The TM values
between the sticky ends of three-point-star motifs were
measured using other DNA sequences that form hairpin
structures (see Experimental Section). In Table 1, hpM&hpS,
hpM-1azo&hpS, hpM&hpS-1azo, hpM-1azo&hpS-1azo, andhpM-2azo&hpS-2azo correspond to the hybridization between the sticky ends of three-point-star motifs formed by
L&M&S, L&M-1azo&S, L&M&S-1azo, L&M-1azo&S-1azo,
and L&M-2azo&S-2azo, respectively. All of the TM values of
the azobenzene-modified strands were higher than that of
the original strands without azobenzenes. This result supported our hypothesis that azobenzenes stabilize DNA structures and was consistent with previous reports.21 Interestingly, the TM of hpM-2azo&hpS-2azo, which contains fourazobenzenes, was higher than that of hpM&hpS by as muchas 6.7 °C, indicating that much more stable capsules wereformed by using L&M-2azo&S-2azo. In fact, the TM of DNA
capsules formed by L&M-2azo&S-2azo was much higher(56.3 °C) than that formed by L&M&S (50.1 °C), although
the melting profiles were not as clear (see Supporting
Information).
The stabilizing effects did not increase in proportion to
the number of azobenzenes. The structures formed by hpM1azo&hpS were much more stable than those formed by
hpM&hpS-1azo, although they had the same number ofazobenzenes. This indicates that the stability of the DNA
capsules was also affected by the position of the azoben© 2010 American Chemical Society

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FIGURE 2. The leftmost figures are AFM images of L&M&S-1azo (upper) and L&M-2azo&S-2azo (lower) in air. The scale bars in the insets are
100 nm. The heights at the lines denoted by (a) and (b) in the AFM images are shown in the two rightmost figures.
TABLE 1. Melting Temperatures (TM) between Two Sticky Ends with or without Azobenzenes
name of hairpin
no. of Azos
TM (°C)

hpM&hpS037.3

hpM-1azo&hpS1
40.4

hpM&hpS-1azo
1
38.5

hpM-2azo&hpS-2azo
4
44.0

TTT CTT GCC AGG CAC CAT CGT AGG TTT CTT GCC AGGCAC CAT CGT AGG TTT CTT GCC; medium strand M, TAG
CAA CCT GCC TGG CAA GCC TAC GAT GGA CAC GGT AAC
GCC; and short peripheral strand S, TTA CCG TGT GGT TGC
TAG GCG. To confirm the formation of a single three-pointstar motif, we prepared a short strand without a sticky end(S-noEnd): TTA CCG TGT GGT TGC TA. DNA sequences
modified with azobenzenes were also prepared: medium
strand with one azobenzene (M-1azo), TAG CAA CCT GCC
TGG CAA GCC TAC GAT GGA CAC GGT AAC GXC C; medium
strand with two azobenzenes (M-2azo), TAG CAA CCT GCC
TGG CAA GCC TAC GAT GGA CAC GGT AAC XGC XC; shortstrand with one azobenzene (S-1azo), TTA CCG TGT GGTTGC TAG GXC G; short strand with two azobenzenes (S2azo), TTA CCG TGT GGT TGC TAG XGC XG, where X
denotes the azobenzene. All azobenzenes were inserted into
the sticky ends of three-point-star motifs.
Sample Preparation. Oligonucleotides without azobenzenes were supplied by Sigma-Aldrich, Japan, and synthesizedusing polyacrylamide gel electrophoresis (PAGE) purification.
Oligonucleotides with azobenzenes were supplied by Tsukuba
Oligo Service and purified by high-performance liquid chromatography (HPLC). All oligonucleotides were dissolved in Trisacetate-EDTA-Mg2+ (TAE/Mg2+) buffer containing 40 mM Trisacetate (pH 8.0), 1 mM EDTA, and 12.5 mM magnesium
acetate. The oligonucleotide concentrations were determinedby the difference in absorbance at 260 and 320 nm using
extinction coefficients calculated from the nucleotides andnumber and position of azobenzenes. In particular, the yieldof DNA capsules formed by L&M&S-1azo was lower than theothers. The upper band of L&M&S-1azo was not clear,indicating that the main product was not a DNA capsule. This
result was consistent with the AFM results, which showed
only a few DNA capsules (Figure 2). The difference in yields
of DNA capsules is probably due to the effect of the positionof azobenzenes on the conformation of DNA capsules. The
stabilization effect of the position of the azobenzenes needs
further analysis.
In conclusion, we successfully used azobenzenes toconstruct robust and photocontrollable DNA capsules. We
showed that the number and position of azobenzenes in thecapsules dramatically affected their stability and sensitivity
to UV light and demonstrated that azobenzenes can stabilize
DNA capsules. These results suggest that using azobenzenerich DNA strands enables us to form DNA capsules that canbe opened at a designated time and place by short exposure
to UV irradiation and that are robust against changes in theenvironment such as temperature and ionic strength. This
strategy is applicable to other DNA motifs such as DX tiles,2cross tiles,4 and five-point-star motifs17 on inserting azobenzenes into the sticky ends. This enhances the potential for
DNA structures to be used in technical applications, such as
drug-delivery systems as carriers.
Experimental Section. Oligonucleotides. The DNA sequences used in this study were the same as those describedby He et al.:16 central long-strand L, AGG CAC CAT CGT AGG© 2010 American Chemical Society

hpM-1azo&hpS-1azo
2
39.2

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FIGURE 3. The upper figures are AFM images of azobenzene-rich DNA capsules (L&M-2azo&S-2azo) using fast-scanning AFM. Scale bars are
100 nm. The lower figures are the heights at the lines denoted by the symbols (a) and (b) in the AFM images.

FIGURE 4. Agarose gel electrophoresis (0.5%) analysis of DNA capsules at 4 °C with different lengths of UV irradiation (shown at the bottomof the gel image). The symbols - and + at the top of the gel image denote without UV irradiation and with UV irradiation, respectively. Single
bands in the leftmost lane in all gels correspond to three-point-star motifs formed by L&M&S-noEnd. Because the sequence S-noEnd did nothave a sticky end, the assembled structure of L&M&S-noEnd remained as a single three-point-star motif. Pairs of lanes from left to rightcorrespond to (1) L&M&S, (2) L&M-1azo&S, (3) L&M&S-1azo, (4) L&M-1azo&S-1azo, and (5) L&M-2azo&S-2azo.

dinucleoside phosphates based on a nearest-neighbor approximation.22 The absorbance was measured at 90 °C on a
Shimadzu UV-1650PC spectrophotometer to prevent the oligonucleotides from forming secondary structures.
© 2010 American Chemical Society

Self-Assembly of DNA Complexes. DNA sequences, L,
R (R is M, M-1azo or M-2azo), and β (β is S, S-1azo or S-2azo),
were mixed at concentrations of 500 nM, 1.5 µM, and 1.5µM, respectively. Under these conditions, three-point-star
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FIGURE 5. Hybridization between two hairpins, hpM-2azo and hpS-2azo. The X denotes azobenzenes.

motifs form buckyballs.16 The mixed solutions were annealed from 95 °C to room temperature over 48 h using a
hot water bath made of Styrofoam. The annealing schedule
was confirmed by measuring the temperature with a datalogger SuperThermochron (KN Laboratories).
AFM. Because AFM images are very sensitive to theexperimental conditions such as probes, we performed theAFM observations carefully using various settings. The images in Figure 2 were obtained under dry conditions in AC
mode on an MFP-3D AFM (Asylum Research) using NCH
probes (NANOSENSORS). A 2 µL drop of DNA solution was
spotted onto a freshly cleaved mica surface and left for 10
min to achieve adsorption. Then the drop was washed twicewith 100 µL of 2 mM magnesium acetate solution and dried
with compressed air. The images in Figure 3 and the movie
in the Supporting Information were obtained under wet
conditions using fast-scanning AFM (Nano Live Vision, RIBM)
using BL-AC10EGS probes (Olympus). The scanning rate was
0.5 frames/s. A 1 µL drop of 2 mM Mg(CH3COO)2 wasspotted onto a freshly cleaved mica surface. Then 1 µL of
DNA solution diluted five times was added and left for 10
min to achieve adsorption. AFM imaging was performed
with an additional 80 µL of 2 mM Mg(CH3COO)2. The image
shown in Supplementary Figure 1 (Supporting Information)
was taken using a 2 µL drop DNA solution spotted onto a
freshly cleaved mica surface with 30 µL of TAE/Mg2+ buffer.
DNA samples were imaged under wet conditions in tapping
mode on a Multimode AFM with a Nanoscope IIIa controller(Veeco) using BL-AC40TS probes (Olympus). The images
shown in Supplementary Figure 2 (Supporting Information)
were obtained under wet conditions using dynamic mode
AFM operated in the frequency modulation detection technique using NCH probes (Nanoworld). The AFM was modified so that it was suited for in-liquid measurements, basedon a commercial AFM system (JEOL: JSPM-5200). A 2 µL
drop of DNA solution was spotted onto a freshly cleaved
mica surface and left for 10 min to achieve adsorption. Then
30 µL of TAE/Mg2+ buffer with 1 µL of 50 mM Ni2+ wasadded. The AFM images in Figure 2 and Figure 3 wereanalyzed using WSxM software.23
TM Measurement. Because three-point-star motifs hybridize to one another cooperatively and form into a large
structure (polyhedron), it is difficult to analyze the hybridization between two sticky ends of three-point-star motifs. To
simplify the measurement of TM between the sticky-ends,
we prepared special sequences that formed hairpin structures: hpM, TTA CCG TTT TCG GTA ACG CC; hpS, TAG CAACTT TTG TTG CTA GGC G; hpM-1azo, TTA CCG TTT TCG GTA© 2010 American Chemical Society

ACG XCC; hpS-1azo, TAG CAA CTT TTG TTG CTA GGX CG;
hpM-2azo, TTA CCG TTT TCG GTA ACX GCX C; and hpS2azo, TAG CAA CTT TTG TTG CTA GXG CXG, where X
denotes the azobenzene. The sticky ends of these hairpin
structures were designed to be the same as the sticky endsof the three-point-star motifs. Consequently, the hairpin
formed by hpM, hpM-1azo, or hpM-2azo further hybridizes
to that formed by hpS, hpS-1azo, or hpS-2azo (see Figure5). This hybridization process reveals a more simple melting
profile than the hybridization of three-point-star motifs.
Thus, this hairpin system was relatively easy to analyze. The
TM values at 2 µM except for that of hpM-2azo&hpS-2azo
were measured at 260 nm in TAE/Mg2+ buffer. For hpM2azo&hpS-2azo, it was difficult to measure the TM at 260 nm,
because it was very close to that of the hairpin structure
itself. Instead, we measured the TM at 320 nm. When two
azobenzenes interact with each other, the absorbance at 320
nm is changed by the interaction. In contrast, the absorbance of DNA sequences at 320 nm is very small and canbe negligible. Thus, the hybridization between two sticky
ends formed by hpM-2azo&hpS-2azo can be monitored by
the absorbance change at 320 nm caused by the interaction
between azobenzenes.
UV Light Irradiation. For gel electrophoresis, UV lightat a wavelength of 365 nm was irradiated using an OMRONZUV-C20H UV-LED irradiator with a ZUV-H10 M head unit
and ZUV-L6T lens, after annealing. To increase the efficiency
of UV irradiation, 10 µL of DNA solution was used. For fastscanning AFM, we used another UV irradiator system thatis optionally supplied with the AFM: a fluorescence mirror
unit (U-MWU2, Olympus), a mercury lamp (USH-1030 L,
Olympus), a mercury lamp housing (U-LH100HGAPO, Olympus), and a power supply unit (U-RFL-T, Olympus).
Agarose Gel Electrophoresis. Gels containing 0.5% agarose were run on a Mupid-exu electrophoresis unit (Advance)
at 4 °C and a constant voltage of 50 V for 75 min. The
running buffer was TAE/Mg2+. After electrophoresis, the gelswere stained with SYBR Gold for 25 min and scanned. The
bands were quantified using the ImageJ 1.43u software(National Institutes of Health, USA).
Acknowledgment. This work was supported by a Grantin-Aid for Scientific Research (B) (20300106) and a grantfrom the Circle for the Promotion of Science and Engineering. We thank Professor Chengde Mao (Purdue University)
for helpful discussions. We also thank Akinori Kuzuya(University of Tokyo), Miho Tagawa (PRESTO, JST), Shogo
Hamada (Titech), and Miki Hirabayashi (NICT) for teaching
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us how to use AFM, and Motoharu Shichiri (RIBM) for taking
some AFM images.

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Supporting Information Available. Detailed structures ofa three-point-star motif and an azobenzene, other AFM
images of the structures formed by L&M&S-1azo or L&M2azo&S-2azo, a melting curve of DNA capsules formed by
L&M&S or L&M-2azo&S-2azo, and the movie file producedby fast-scanning AFM, which clearly demonstrated how the
DNA capsules formed by L&M-2azo&S-2azo broke down.
This material is available free of charge via the Internet at
http://pubs.acs.org.

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