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Stabilization of DNA nanostructures by photo-cross-linking†

Miho Tagawa,‡*ab Koh-ichiroh Shohda,a Kenzo Fujimotoc and Akira Suyamaa

Received 11th July 2011, Accepted 2nd September 2011

DOI: 10.1039/c1sm06303k

Developing methods for stabilizing DNA nanostructures is a major challenge for next-generation

nanofabrication, because stable DNA nanostructures are expected to work as building materials in the

bottom-up assembly of functional biomolecules and nano-electronic components. Here we show the

availability of cross-linking-type photoreaction with 3-cyanovinylcarbazole nucleosides (CNVKs) on

DNA nanostructures. DNA double-crossover AB-staggered (DXAB) tiles including cross-linking

molecules, CNVKs, self-assembled into two-dimensional (2D) periodic DNA arrays and were covalently

connected by photo-cross-linking. The self-assembled DNA arrays before and after photo-cross-

linking have been visualized by high-resolution, tapping mode atomic force microscopy (AFM) in

buffer. The improvement of the heat tolerance of photo-cross-linked DNA arrays was confirmed by

heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the

potential of DNA as a functional material in biotechnology and nanotechnology.

Introduction

The greatest benefits in using DNA as an engineering material lie

in its ability to self-assemble, and its programmable molecular-

recognition. Recently, DNA has been recognized as a useful

building material for nanotechnology,1–5 and great progress has

been made in generating self-assembled periodic DNA arrays

with nanometre-scale precision,6 patterned structures,7–9 three-

dimensional shapes10–12 and nano-mechanical devices.13–15 Thus,

these DNA nanostructures are expected to be available as scaf-

folds for next-generation nanofabrication. However, as engi-

neering materials, the DNA scaffolds are unsuitable because they

are floppy, weak, fragile, and heat-labile. Therefore, it is difficult,

in manipulating DNA nanostructures, via external force and

repeated thermal changes, to control the positioning of various

nano-components with nanometre-scale precision. Enzymatic

nick-ligation of DNA strands is ineffective to stabilize DNA

nanostructures, because enzymes cannot access the extremely

tight space on the nanostructures where DNA strands are

entangled tightly and intricately. Therefore, non-enzymatic

reactions are needed for stabilizing DNA nanostructures.

aDepartment of Life Sciences and Institute of Physics, Graduate School ofArts and Sciences, The University of Tokyo, Komaba, 153-8902, Japan.E-mail: suyama@dna.c.u-tokyo.ac.jp; Fax: +81 5454 6528; Tel: +815454 6528bJapan Science and Technology Agency (JST) PRESTO, JapancSchool of Material Science, Japan Advanced Institute of Science andTechnology, Ishikawa, 923-1292, Japan

† Electronic supplementary information (ESI) available. See DOI:10.1039/c1sm06303k

‡ Present address: Center for Functional Nanomaterials, BrookhavenNational Laboratory, Upton, NY, 11973, USA. Tel: +1 631 344 4812;mtagawa@bnl.gov

This journal is ª The Royal Society of Chemistry 2011

We previously developed heat-resistant DNA arrays stabilized

by 5-carboxyvinyl-20-deoxyuridine (CVU), with which we photo-

ligated the DNA nicks, and improved the heat resistance of the

arrays (Fig. 1a and S1†).16,17 However, stabilization of DNA

duplexes with photoreaction is not limited to photoligation. The

Fig. 1 Design of a DXAB tile and its arrangement into a 2D DNA

array. (a) Photoligation with CVU. (b) Photo-cross-linking with CNVK. (c)

The sequences of the DXAB tile for constructing photo-cross-linked 2D

DNA arrays. Ks represent CNVK bases. Arrowheads at the ends of strands

indicate the 30-terminals. The solid rhomboids at the 50-ends represent

phosphorylation. (d) The lattice topology of a 2D DNA array produced

by the DXAB tiles. The solid circles represent CNVK bases.

Soft Matter, 2011, 7, 10931–10934 | 10931

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cross-linking-type photoreaction is another effective method and

can stabilize the DNA duplex more tightly than photoligation

due to its structural stability.

The most popular cross-linking molecule used to stabilize

DNA duplexes is psoralen.18 However, cross-linking with

psoralen is not suitable for stabilizing DNA nanostructures

because it involves the integration of another molecule that

engenders a large deformation in the DNA duplex. In contrast,

photo-cross-linking with 3-cyanovinylcarbazole nucleosides

(CNVKs)19 does not involve any additional molecule becauseCNVK is first introduced into the DNA strand, and therefore it

may be a promising method to tightly stabilize DNA nano-

structures with small deformations. CNVK included in the

modified oligodeoxynucleotide binds to an adjacent pyrimidine

base in the complementary strand via 366 nm UV irradiation

(Fig. 1b). When the bonding partner is a thymine base, the

efficiency of photo-cross-linking with CNVK is considerably

high, about 97%, in the various sequence sets.19 Thus, the

DNA photo-cross-linking using CNVKs has great potential for

tightly stabilizing DNA nanostructures with high yield without

large structural deformations. Herein, we report our attain-

ment of DNA arrays stabilized by the photo-cross-linking

with CNVKs.

Fig. 2 AFM images and section profiles of non-UV-exposed 2D DNA

arrays of DXAB tiles before or after heat treatment on mica surfaces

under buffer droplets for 2 min. The scan sizes of images are 1 mm� 1 mm

for large images, and 150 nm � 150 nm for the inset images. The section

profiles correspond to the white lines in the inset images. The vertical axis

of the section profile in (a) is drawn on a different scale from the others.

(a) Non-UV-exposed DNA arrays without heat treatment. Non-UV-

exposed DNA arrays after heating at 40 �C (b), 45 �C (c), and 50 �C (d).

Results

DNA arrays including CNVKs

The DXAB tile, which is used to construct 2D DNA arrays by

self-assembly, consists of two parts, A and B, derived from the

well-known DX tiles.20 Parts A and B are held together by

strand-ab, and, due to the nicks, their junction point has a certain

degree of flexibility. We designed the sequences of sticky ends

specifically to carry out the photo-cross-linking reactions. The 50-ends of strand-a2 and strand-b2 have UV-sensitive CNVKs that

bind to the thymine bases diagonally opposite under 366 nm UV

irradiation (Fig. 1c). The complementary sticky-end pairs bind to

each other, so that the DXAB tiles form the photo-cross-linked

2D arrays (Fig. 1d).

The self-assembled DXAB tile arrays before and after photo-

cross-linking were visualized by atomic-force microscopy

(AFM). The samples were deposited for adsorption on atomi-

cally flat mica surfaces and then imaged in 1� TAE/Mg2+ buffer.

The DXAB tile arrays before photo-cross-linking had a micro-

scale periodic structure (Fig. 2a). The average distance of the

longitudinal AB period (the long-axis period) was about 30 nm,

in good agreement with the designed parameters. The distance of

the short-axis period measured from section profiles differed

from area to area, from about 5 nm to 8 nm. The difference in

elevation of the periodic corrugation also varied from 0.25 nm to

4 nm, values that are correspondingly shorter and longer than the

diameter of a hydrated DNA duplex (2 nm).

After UV-ray exposure, we acquired the images of the arrays

after a couple of scans in the same area (Fig. 3a). The photo-

cross-linked arrays show almost the same periodic structure as

some areas of the array before photoreaction (150 nm � 150 nm

image in Fig. 3a), with the long- and short-axis periods of about

30 nm and 5 nm, respectively; however, resolution is insufficient

to measure accurately the periodicity of the arrays.

10932 | Soft Matter, 2011, 7, 10931–10934

Heat-resistance of photo-cross-linked DNA arrays

We heated the 2D DNA arrays before and after photo-cross-

linking in adsorbed states on mica surfaces under buffer droplets

to quantify the effect of photo-cross-linking on the heat toler-

ance, and to check the structural changes occurring during

annealing. After heat treatment, samples were left at room

temperature for cooling, and then imaged at room temperature.

Fig. 2b–d show AFM images of non-UV-exposed arrays after

heating at 40 �C, 45 �C, and 50 �C, respectively, for 2 min;

Fig. 3b–f show AFM images of UV-exposed arrays after heating

at 55 �C, 60 �C, 65 �C, 70 �C and 75 �C, respectively, for 2 min.

After heat treatment, we were able to image the photo-cross-

linked DNA arrays at first scans without continuously tapping

them with the AFM tip. In addition, from the well-ordered

periodic array images in Fig. 3c–e, we reliably confirmed that no

major conformational changes occurred after UV-ray exposure

in buffer solutions. The long and short axis periods were about

This journal is ª The Royal Society of Chemistry 2011

Fig. 3 AFM images and section profiles of UV-exposed 2DDNA arrays

before or after heat treatment on mica surfaces under buffer droplets

for 2 min. The scan sizes of images are 1 mm� 1 mm for large images, and

150 nm � 150 nm for inset images. The section profiles correspond to the

white lines in the inset images. The vertical axis of the section profile in (a)

is drawn on a different scale from others. (a) UV-exposed DNA arrays

without heat treatment. UV-exposed DNA arrays after heating at 55 �C(b), 60 �C (c), 65 �C (d), 70 �C (e), and 75 �C (f).

This journal is ª The Royal Society of Chemistry 2011

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30 nm and 7 nm, respectively, in high-resolution images after

heating at 70 �C (Fig. 3e).

The non-UV-exposed arrays maintained their form after

heating at 40 �C (Fig. 2b), but barely maintained it after heating

at 45 �C, exhibiting many defects (Fig. 2c). After heating at

50 �C, the arrays were broken up completely and periodic arrays

were not apparent (Fig. 2d). In contrast, UV-exposed arrays

were not disrupted after heating at 55 �C, 60 �C, 65 �C and 70 �C(Fig. 3b–e). Periodic arrays were no longer observed clearly after

heating to 75 �C, and the section profile showed disordered

structures (Fig. 3f), indicating they were beginning to break-

down. We confirmed that it is safe to heat arrays at 70 �C for

2 min to keep ordered arrays. Therefore, their heat tolerance is

sufficient practically for binding or removing other molecules or

particles. The DNA arrays were stabilized by the photo-cross-

linking and their heatproof temperature was improved about

10 �C compared with previously developed photoligated DNA

arrays using CVU.16 This difference may be due to their different

reaction types, although the number of photoreaction sites per

single DNA tile is the same. Due to cross-linking between two

complementary strands, the tiles can be connected more firmly

compared with those photoligated; consequently, the heatproof

temperature of the arrays increased.

The effect of heat treatment as annealing on photo-cross-linked

DNA arrays

We also looked into the effect of heat treatment as annealing on

the structure of DNA arrays. As we increased the annealing

temperature, images of UV-exposed arrays became clearer

(Fig. 3b–e), whereas, in contrast, the images of non-UV-exposed

arrays became fainter (Fig. 2b and c). The lattice of UV-exposed

arrays appeared to be rearranged in a more orderly fashion than

that before annealing (Fig. 3c–e); even the connections of the

sticky ends were visible (inset, Fig. 3e). Further, the section

profiles after annealing showed lower corrugations than the ones

before annealing, with or without photoreactions (Fig. 2 and 3).

This may be because the arrays adsorbed tightly to the mica

surface and DNA duplexes consisting of DNA arrays flattened

by adsorption force, or the tip could not touch the mica surface

through the narrow interspaces between DNA duplexes of close-

packed DNA arrays after annealing. These results show that

annealing at higher temperature available for photo-cross-linked

arrays is effective to obtain 2D DNA arrays with more ordered

structures.

Discussion

We have demonstrated the availability of photo-cross-linking

reaction on DNA nanostructures. The self-assembly of DXAB

tiles including CNVKs generates 2D periodic arrays, and after

photo-cross-linking reactions the arrays maintained their forms

without breakup or remarkable conformational changes. The

heat resistance of the self-assembled DNA arrays improved at

least 30 �C by the photo-cross-linking and we showed photo-

cross-linking using CNVKs is more effective than photoligation

using CVUs for stabilization of DNA tile arrays. Therefore, we

believe that the photo-cross-linking reaction using CNVKs is

another promising method to stabilize complexes with DNA by

Soft Matter, 2011, 7, 10931–10934 | 10933

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a covalent-bond formation with high reactivity, affording huge

potential for use in DNA nanotechnology. The stabilized DNA

scaffolds maintain the address space even in solution and under

high temperature, so that nano-components are positioned with

nanometre-scale precision. The scaffolds can be applied to

manufacturing nano-electronic devices using manipulation

methods such as probe manipulation or optical tweezers and

heating processes. Non-crushed, rigid DNA containers with

address space can be assembled using this photo-cross-linking

and DNA self-assembly, and might well serve as nano-scale

reaction vessels to encompass selectively various molecules and

nanoparticles. Similarly, we might construct 3D complex struc-

tures by the stepwise assembly of rigid, heat-resistant DNA

components. Branched DNA strands created by photoreaction

will facilitate the building of substantially smaller DNA tiles and

more complex 3D DNA nanostructures, which could be used

advantageously in the fields of biotechnology and

nanotechnology.

Experimental

DNA sequences, synthesis and purification

The DNA strand sequences of the DXAB tile were based on the

DXAB tile previously designed,16 and modified to carry out

photo-cross-linking reaction. The strands also were designed to

have no stable self-folded structures that prevent planned

hybridization. The stability of self-folded structures of the

strands was checked by mfold21,22 and HyFol (http://www.

nanobiophys-sakura.net/HyFol/index.html). The DNA strand

sequences of the DXAB tile are shown in Fig. 1c. All DNA

strands in this study were synthesized and purified by HPLC or

PAGE commercially (Nihon Gene Research Laboratories Inc.).

Strands containing CNVKs were synthesized using the phosphor-

amidite of CNVK that was synthesized according to the previously

reported method.19

Annealing of DNA strands of DNA tile arrays

Sets of strands for constructing the DXAB tiles were mixed

stoichiometrically and dissolved to 0.8 mM in 1� TAE/Mg2+

buffer (40 mM Tris-acetate, 1 mM EDTA, 12.5 mM Mg acetate,

pH 8.3). The solutions were annealed from 95 �C to 45 �C for 3 h,

and thereafter from 45 �C to 26 �C for 13 h in a Peltier Thermal

Cycler PTC-200 (MJ Research Inc.). During the first annealing

process, DNA single strands were grouped into DXAB tiles;

during the second process, the tiles were assembled into 2DDNA

arrays through complementary base sequences in their sticky

ends.

Photo-cross-linking reactions

For photo-cross-linking, an aliquot of the annealed array solu-

tion in another tube was exposed to 366 nm UV-rays on ice for

2 min using an UV-LED illuminator ZUV-C10 (OMRON Inc.).

AFM imaging

For AFM imaging in buffer, a 4 ml sample drop was spotted on

freshly cleaved mica (Nihon Shoji Co., Ltd.) and left to adsorb to

10934 | Soft Matter, 2011, 7, 10931–10934

the surface for 3 min. Then, 22 ml of 1� TAE/Mg2+ buffer was

placed onto the mica and another 22 ml of the buffer was pipetted

onto the AFM tip. Atomic force micrographs were obtained in

tapping mode in buffer at room temperature on a NanoScope V

(Digital Instruments) equipped with a multimode head with

BioLever mini tips (Olympus Inc.).

Heat treatment

For heat treatment, the self-assembled 2D DNA arrays before

and after UV exposure were heated in the adsorbed state on the

mica surfaces under 30 ml buffer droplets on a Thermo Block

ND-M01 (NISSIN Inc.) at 100% relative humidity to prevent

drying. The heated samples were left at room temperature for

cooling before AFM imaging.

Acknowledgements

This work was partly supported by PRESTO-JST, Grants-in-Aid

for Scientific Research in Priority Areas ‘Search of Nanosystems

and Function Emergence’ to MT and for Scientific Research (A)

20241029 to AS from theMinistry of Education, Culture, Sports,

Science, and Technology, Japan. The authors gratefully

acknowledge Avril Woodhead (BNL) for the manuscript editing.

Notes and references

1 N. C. Seeman, Nature, 2003, 421, 427–431.2 A. J. Turberfield, Phys. World, 2005, 16, 43–46.3 Y. He, H. Liu, Y. Chen, Y. Tian, Z. Deng, S. H. Ko, T. Ye andC. Mao, Microsc. Res. Tech., 2007, 70, 522–529.

4 H. Yan, Science, 2004, 306, 2048–2049.5 C. Steinhauer, R. Jungmann, T. L. Sobey, F. C. Simmel andP. Tinnefeld, Angew. Chem., 2009, 121, 9030–9034; Angew. Chem.,Int. Ed., 2009, 48, 8870–8873.

6 E. Winfree, F. Liu, L. A. Wenzler and N. C. Seeman, Nature, 1998,394, 539–544.

7 P. W. Rothemund, Nature, 2006, 440, 297–302.8 S. H. Park, C. Pistol, S. J. Ahn, J. H. Reif, A. R. Lebeck, C. Dwyerand T. H. LaBean, Angew. Chem., 2006, 118, 749–753; Angew.Chem., Int. Ed. 2006, 45, 735–739.

9 H. Yan, T. H. LaBean, L. Feng and J. H. Reif, Proc. Natl. Acad. Sci.U. S. A., 2003, 100, 8103–8108.

10 Y. He, T. Ye, M. Su, C. Zhang, A. E. Ribbe, W. Jiang and C. Mao,Nature, 2008, 452, 198–201.

11 F. C. Simmel, Angew. Chem., 2008, 120, 5968–5971; Angew. Chem.,Int. Ed. 2008, 47, 5884–5887.

12 S. M. Douglas, H. Dietz, T. Liedl, B. Hogberg, F. Graf andW. M. Shih, Nature, 2009, 459, 414–418.

13 R. P. Goodman, M. Heilemannt, S. Dooset, C. M. Erben,A. N. Kapanidis and A. J. Turberfield, Nat. Nanotechnol., 2008, 3,93–96.

14 H. Yan, X. Zhang, Z. Shen and N. C. Seeman, Nature, 2002, 415, 62–65.

15 L. Feng, S. H. Park, J. H. Reif and H. Yan, Angew. Chem., 2003, 115,4478–4482; Angew. Chem., Int. Ed. 2003, 42, 4342–4346.

16 M. Tagawa, K. Shohda, K. Fujimoto, T. Sugawara and A. Suyama,Nucleic Acids Res., 2007, 35, e140.

17 Y. Yoshimura, Y. Noguchi, H. Sato and K. Fujimoto,ChemBioChem, 2006, 7, 598–601.

18 H. P. Spielmann, T. J. Dwyer, J. E. Hearst and D. E. Wemmer,Biochemistry, 1995, 34, 12937–12953.

19 Y. Yoshimura and K. Fujimoto, Org. Lett., 2008, 10, 3227–3230.20 T. J. Fu and N. C. Seeman, Biochemistry, 1993, 32, 3211–3220.21 D. H. Mathews, J. Sabina, M. Zuker and D. H. Turner, J. Mol. Biol.,

1999, 288, 911.22 M. Zuker, Nucleic Acids Res., 2003, 31, 3406–3415.

This journal is ª The Royal Society of Chemistry 2011