A photon-fueled DNA nanodevice that contains two different photoswitches

Article abstract of DOI:10.1002/anie.201106093

DNA seesaw: Photoswitchable azobenzenecarboxylic acid 1 reversibly photoisomerizes between the trans form and the thermally stable cis form upon irradiation with visible light. A photon-fueled DNA nanodevice that moves like a seesaw in response to irradia

Full text of DOI:10.1002/anie.201106093

Angewandte
Chemie
DOI: 10.1002/anie.201106093
Molecular Devices
A Photon-Fueled DNA Nanodevice that Contains Two Different
Photoswitches**
Hidenori Nishioka, Xingguo Liang,* Tomohiro Kato, and Hiroyuki Asanuma*
DNA is a promising, self-assembling nanomaterial for the
construction of delicate nanostructures and nanodevices.[1–5]
However, when short DNA oligonucleotides are used as the
fuel to drive a nanodevice, the waste gradually accumulates
and lowers the operating efficiency.[3,4] Accordingly, efforts
have been made to construct new nanodevices that use
“clean” fuels. For example, Liu and co-workers utilized
electric signals to reversibly drive a DNA switch.[6] We
constructed photon-fueled nanomachines that did not dete-
riorate after many working cycles.[7]
A photon-fueled DNA nanodevice has been constructed
based on the reversible photoregulation of DNA hybrid-
ization in azobenzene-modified DNA. The planar trans-
azobenzene intercalates between adjacent base pairs and
stabilizes the duplex through stacking interactions, whereas
the nonplanar cis-azobenzene destabilizes the duplex by steric
hindrance.[8,9] To date, photoswitches have essentially con-
tained a single species, such as azobenzene-4’-carboxylic acid
(Azo) or 2,6-dimethylazobenzene-4’-carboxylic acid (DMazo;
Scheme 1). Ultraviolet (310–370 nm) or visible light (l
greater than 400 nm) induces reversible isomerization
between the trans and cis forms of such compounds. We
sought to develop a photoswitch that photoisomerizes upon
irradiation at other wavelengths to diversify the photo-
Scheme 1. Structures of azobenzene derivatives introduced into DNA.
switches and to make them applicable for various purposes
where UV light should be avoided. In this study, we
synthesized an azobenzene derivative that photoisomerizes
reversibly upon irradiation with visible light. When tethered
onto d-threoninol and linked to DNA, 2,6-dimethyl-4-(meth-
ylthio)azobenzene-4’-carboxylic acid (S-DMazo, Scheme 1)
had a sufficiently high cis content, acceptable thermal
stability, and a high photoregulatory efficiency. By combining
Azo and S-DMazo, a photon-fueled DNA nanomachine that
The sequence of a modified DNA (CXG) and its complementary
sequence (GC) used in the model system of photoregulation of DNA
hybridization are also shown.
moved with a seesaw-like motion upon irradiation with light
of different wavelengths was produced.
The trans isomers of azobenzene and its alkyl derivatives
have absorption maxima (lmax) at around 320 nm (p–p*
transition), and the cis-rich form can be obtained by
irradiation with light of wavelengths between 310–
370 nm.[10] An azobenzene derivative with a large bathochro-
mic shift to the visible region can be obtained by the
introduction of an electron-donating group at the para
position. However, the overlap of p–p* and n–p* transitions
(at ca. 450 nm) makes it difficult to obtain the cis-rich form,
and the stronger conjugation as well as the electronic effects
caused by these substituents decrease the thermal stability of
the cis form.[11] To avoid large overlap of the p–p* and n–p*
transitions in the trans form and to ensure a high thermal
stability in the cis form, the ideal azobenzene derivative
would have a lmax at around 400 nm. We designed S-DMazo to
satisfy these requirements. In S-DMazo, the methylthio group
at the para position of the distal benzene ring causes a
bathochromic shift in the lmax value of the trans form to
400 nm.[12] The two methyl groups at the ortho positions of the
distal ring enhance the photoregulatory efficiency and the
[*] Dr. H. Nishioka, Prof. Dr. X. Liang, T. Kato, Prof. Dr. H. Asanuma
Department of Molecular Design and Engineering
Graduate School of Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)
E-mail: liang@mol.nagoya-u.ac.jp
asanuma@mol.nagoya-u.ac.jp
Prof. Dr. X. Liang
School of Food Science and Engineering
Ocean University of China
Yushan-lu 5, Shinanqu, Qingdao 266003 (P.R. China)
E-mail: liangxg@ouc.edu.cn
[**] This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology (Japan, nos. 20750132, 21241031, and 18001001).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106093.
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Table 1: Tm values for duplexes CXG/GC that contain various azoben-
zene derivatives and the thermal stability of their cis forms.[a]
Azobenzene
derivative (R)
Tm [8C][b]
cis
t1/2 of cis [h][c]
[d]
trans
DTm
S-DMazo
Azo[e]
48.3
48.9
46.1
50.9
34.9
43.2
45.2
36.3
13.4
5.7
0.9
6.4
3.3
0.42
25
S-Azo
DMazo[e]
14.6
[a] Solution conditions: DNA (5 mm), NaCl (100 mm), phosphate buffer
(10 mm, pH 7.0). [b] Tm value of the unmodified DNA duplex is 47.78C.
[c] Half-lives of cis-azobenzene derivatives for the thermal isomerization
at 608C. [d] Change of Tm value induced by cis–trans isomerization. [e] For
comparison, data for Azo and DMazo duplexes are also listed.
13.48C (see Figure S3 in the Supporting Information for the
actual melting curves). This difference is twofold larger than
that of the corresponding Azo-modified duplexes. For this
experiment, the trans-to-cis and cis-to-trans photoisomeriza-
tions were carried out at 400 nm and 450 nm, respectively;
thus, the photoregulation of DNA hybridization with only
visible light was achieved. The two methyl groups at the ortho
positions of the distal ring were essential as the duplex that
was modified with S-Azo had a DTm value of only 0.98C. In
contrast, DMazo, without the methylthio group, had a DTm of
14.68C.[13b] The steric bulk of the methylthio group in the para
Figure 1. Absorption spectra of S-DMazo (thick lines) and Azo (thin
lines) tethered onto CXG in either the trans form (solid lines) or in the
cis-rich state (dotted line). Conditions: CXG (20 mm), NaCl (100 mm),
phosphate buffer (10 mm, pH 7.0), 608C. Wavelengths of light for
isomerization of S-DMazo and Azo to their cis forms are 400 nm and
340 nm, respectively. The lmax values for trans-S-DMazo and trans-Azo
are 382 nm and 330 nm, respectively.
thermal stability of the cis form.[13] We introduced S-DMazo
with a d-threoninol linker into the DNA sequence CXG
(Scheme 1) by using standard phosphoramidite chemistry
(Schemes S1 and S2 in the Supporting
Information).[8b] For comparison, Azo,
DMazo, and 4-(methylthio)azobenzene-
4’-carboxylic acid (S-Azo) were also intro-
duced into CXG.
Figure 1 shows the UV/Vis spectra
(recorded at 608C) of Azo- and S-
DMazo-modified CXG in the absence of
its
complementary
sequence
GC
(Scheme 1). The lmax of trans-S-DMazo
was 382 nm, which is 52 nm higher than
that of trans-Azo (lmax, 330 nm). At 258C,
the lmax shifted to 391 nm, and shifted
further to 397 nm when CXG was hybrid-
ized to GC at 258C (Figures S1 and S2 in
the Supporting Information). Upon irra-
diation at 400 nm, 66% of trans-S-DMazo
isomerized to the cis form (Figure 1).
Upon irradiation of trans-S-DMazo at
370 nm, 390 nm, 450 nm, or 520 nm, the
cis content was 66%, 66%, 17%, and
25%, respectively (Table S1 in the Sup-
porting Information). Interestingly, trans-
rich S-DMazo (58% trans form) was
obtained after irradiation at 340 nm.
We evaluated the photoregulatory effi-
ciency of S-DMazo by monitoring the
change in the melting temperature (DTm)
of modified DNA duplexes after the trans–
cis isomerization. As shown in Table 1, the
difference in the melting temperature
between the CXG/GC duplexes that con-
Figure 2. a) Illustration of the design of a DNA nanodevice that contains S-DMazo- and Azo-
modified DNAs. b) Four possible states (a, b, c, and d) of the nanodevice are achieved by
irradiation at four wavelengths (340 nm, 370 nm, 390 nm, and 450 nm, respectively). The 5’
and 3’ termini of the long oligonucleotide (Temp) are modified with fluorophores FAM and
Py, respectively. The oligonucleotide M bar hybridizes with the central region of Temp. Dab is
attached at the 3’ end of the Azo-modified oligonucleotide R bar, and An is attached at the
tain the cis and trans forms of S-DMazo is 5’ end of the S-DMazo-modified oligonucleotide L bar.
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position in the S-Azo-modified duplexes hindered the hybrid-
ization of CXG with the complementary strand. The duplex
with trans-S-DMazo had a Tm of 48.38C, which is comparable
to the native DNA duplex with the same sequence (Tm =
47.78C, Table 1) and is important for constructing stable
nanostructures.
We examined the thermal stability of cis-S-DMazo. The
half-life of cis-S-Azo was approximately 25 min at 608C,
which is much shorter than the half-life of cis-Azo
(ca. 200 min), because the electron-donating methylthio
group greatly reduces the thermal stability of the cis form
(Table 1). Although trans-S-Azo could also be isomerized to
the cis form with 400 nm light (Figure S4 in the Supporting
Information), neither the thermal stability nor the photo-
regulatory efficiency was acceptable for actual applications.
Interestingly, the half-life of cis-S-DMazo was twofold longer
than that of cis-Azo (Table 1), which shows that the presence
of two methyl groups greatly improves the thermal stability of
cis-S-DMazo.[13b]
By combining S-DMazo and Azo, a light-driven DNA
nanodevice that moves like a seesaw was designed. As shown
in Figure 2A, the DNA nanodevice consists of four oligonu-
cleotides: a 20 nucleotide (nt) oligonucleotide that contains
five S-DMazo residues (L bar); a 25 nt unmodified DNA
(M bar); a 20 nt oligonucleotide modified with five Azo
residues (R bar); and Temp, a 65 nt DNA. The working
principle of the nanodevice is illustrated in Figure 2B. After
irradiation at 450 nm, both cis-S-DMazo and cis-Azo isomer-
ize to the trans forms so that both the L bar/Temp and the
R bar/Temp duplexes are formed (state a). Upon irradiation
of the device at 390 nm (or 400 nm), trans-S-DMazo isomer-
izes to the cis form and Azo remains in the trans form so that
the L bar/Temp duplex dissociates and the R bar/Temp
duplex remains stable (state b). After irradiation at 340 nm,
cis-S-DMazo isomerizes to the trans form and trans-Azo
isomerizes to the cis form so that the L bar/Temp duplex
reforms and the R bar/Temp duplex dissociates (state c).
Upon irradiation at 370 nm, trans-Azo and trans-S-DMazo
both isomerize to the cis forms and both L bar/Temp and
R bar/Temp duplexes dissociate (state d). The native duplex
in the middle (M bar/Temp) is a rigid spacer that is a stable
duplex in all states. Manipulation of this DNA nanodevice is
carried out simply by irradiating with the appropriate wave-
lengths of light.
Figure 3. Quantitative analysis of the manipulation of the DNA nano-
device by monitoring the fluorescence of Py (370–450 nm) and FAM
(500–560 nm) after irradiation with the indicated wavelengths of light.
Conditions: DNA (0.1 mm), NaCl (100 mm), phosphate buffer (10 mm,
pH 8.0), 47.58C. Before fluorescence measurements, samples were
irradiated for 15 min at 47.58C.
or 340 nm, which indicated that the R bar/Temp duplex was
dissociated. The L bar/Temp duplex formed after irradiation
at 340 nm or 450 nm, whereas irradiation at 370 nm or 390 nm
caused the L bar/Temp duplex to dissociate. The fluorescence
changes are consistent with the results of the photoisomeri-
zation experiments (Figure 1, see also Tables S1 and S2 in the
Supporting Information) and Tm measurements (Table 1, see
also Table S3 in the Supporting Information). In these
experiments, all irradiations with light were carried out at
47.58C, the same temperature that was used during the
measurements of the fluorescence.[15] These results indicate
that all four states of the DNA nanodevice shown in
Figure 2B were obtained.
The photoregulatory efficiency of S-DMazo was also
quantitatively evaluated by analyzing the changes in the
fluorescence (Figure 3). For example, after irradiation at
340 nm, 60% of the L bar strands were in the duplex form.
After irradiation at 390 nm, 79% of the L bar strands were
dissociated and 94% of the R bar strands were in the duplex
form. Furthermore, repetitive switching between state b and
state c was achieved by alternately irradiating the system at
340 nm and 390 nm. As shown in Figure 4, irradiation at
340 nm caused the R bar/Temp to dissociate and the L bar/
Temp duplex to form, whereas irradiation at 390 nm induced
the dissociation of the L bar/Temp duplex and formation of
the R bar/Temp duplex. Thus, seesaw-like movement was
successfully achieved by irradiation with light (see Figure S5
in the Supporting Information for polyacrylamide gel electro-
phoresis (PAGE) data).
Two fluorophore/quencher systems, pyrene/anthraqui-
none (Py/An)[14] and (6-fluorescein-6-carboxamido)hexa-
noate/4-dimethylaminoazobenzene-4’-carboxylic acid (FAM/
Dab, Figure 2), were used to quantitatively analyze the
hybridization of the duplexes. The two fluorophores (FAM
and Py) were attached to each end of the Temp sequence
(Figure 2). The quenchers An and Dab were attached at the
5’ end of L bar and at the 3’ end of R bar, respectively. The
formation of the duplexes that consist of L bar/Temp and
R bar/Temp should quench emissions from FAM (emission at
500–560 nm) and Py (emission at 370–450 nm), respectively.
As shown in Figure 3, the fluorescence intensity of FAM
decreased after irradiation at 390 nm or 450 nm, which
indicated that the R bar/Temp duplex was formed. The
fluorescence of FAM increased after irradiation at 370 nm
In conclusion, the hybridization of DNA can be efficiently
photoregulated by visible light with the introduction S-
DMazo into an oligonucleotide. Trans-S-DMazo isomerizes
efficiently to the cis form upon irradiation at 400 nm, and the
thermal stability of cis-S-DMazo was higher than that of cis-
Azo as a result of two methyl groups at the ortho positions of
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d) T. J. Bandy, A. Brewer, J. R. Burns, G. Marth, T. Nguyen, E.
Stulz, Chem. Soc. Rev. 2011, 40, 138 – 148.
[2] a) C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff,
Nature 1996, 382, 607 – 609; b) E. Winfree, F. Liu, L. A. Wenzler,
N. C. Seeman, Nature 1998, 394, 539 – 544; c) W. M. Shih, J. D.
Quispe, G. F. Joyce, Nature 2004, 427, 618 – 621; d) P. W. K.
Rothemund, Nature 2006, 440, 297 – 302.
[3] a) B. Yurke, A. J. Turberfield, A. P. Mills, F. C. Simmel, J. L.
Neumann, Nature 2000, 406, 605 – 608; b) T. Liedl, T. L. Sobey,
F. C. Simmel, Nano Today 2007, 2, 36 – 41.
[4] a) Y. He, M. Su, C. Zhang, A. E. Ribbe, W. Jiang, C. Mao, Nature
2008, 452, 198 – 202; b) S. Modi, M. G. Swetha, D. Goswami,
G. D. Gupta, S. Mayor, Y. Krishnan, Nat. Nanotechnol. 2009, 4,
325 – 330.
[5] M. Endo, H. Sugiyama, ChemBioChem 2009, 10, 2420 – 2443.
[6] Y. Yang, G. Liu, H. J. Liu, D. Li, C. H. Fan, D. S. Liu, Nano Lett.
2010, 10, 1393 – 1397.
[7] a) X. G. Liang, H. Nishioka, N. Takenaka, H. Asanuma,
ChemBioChem 2008, 9, 702 – 705; b) M. G. Zhou, X. G. Liang,
T. Mochizuki, H. Asanuma, Angew. Chem. 2010, 122, 2213 –
2216; Angew. Chem. Int. Ed. 2010, 49, 2167 – 2170.
[8] a) H. Asanuma, T. Ito, T. Yoshida, X. G. Liang, M. Komiyama,
Angew. Chem. 1999, 111, 2547 – 2549; Angew. Chem. Int. Ed.
1999, 38, 2393 – 2395; b) H. Asanuma, X. G. Liang, H. Nishioka,
D. Matsunaga, M. Liu, M. Komiyama, Nat. Protoc. 2007, 2, 203 –
212.
[9] a) S. Uno, C. Dohno, H. Bittermann, V. L. Malinovskii, R.
Hꢀner, K. Nakatani, Angew. Chem. 2009, 121, 7498 – 7501;
Angew. Chem. Int. Ed. 2009, 48, 7362 – 7365; b) X. G. Liang, T.
Mochizuki, H. Asanuma, Small 2009, 5, 1761 – 1768; c) X. G.
Liang, H. Nishioka, T. Mochizuki, H. Asanuma, J. Mater. Chem.
2010, 20, 575 – 581; d) F. Tanaka, T. Mochizuki, X. G. Liang, H.
Asanuma, S. Tanaka, K. Suzuki, S. Kitamura, A. Nishikawa, K.
Ui-Tei, M. Hagiya, Nano Lett. 2010, 10, 3560.
[10] G. Zimmerman, L. Y. Chow, U. J. Paik, J. Chem. Phys. 1958, 80,
3528 – 3531.
[11] a) N. Nishimura, T. Sueyoshi, H. Yamanaka, E. Imai, S.
Yamamoto, S. Hasegawa, Bull. Chem. Soc. Jpn. 1976, 49,
1381 – 1387; b) T. Asano, T. Okada, J. Org. Chem. 1986, 51,
4454 – 4458.
Figure 4. Repetitive seesaw-like movement of the DNA nanodevice
between state b and state c induced by alternating light irradiation at
340 nm and 390 nm. Ratios of dissociated L bar/Temp (black circles)
and R bar/Temp (white circles) are shown. Conditions: DNA (0.1 mm),
NaCl (100 mm), phosphate buffer (10 mm, pH 8.0), 47.58C. Each
irradiation was 10 min long.
the distal ring. By combining Azo and S-DMazo, a DNA
nanodevice with seesaw-like movement that is driven by light
was constructed. These results demonstrate that DNA that is
modified with azobenzene derivatives is an excellent nano-
material with which to build elaborate nanodevices. Because
UV light shorter than 380 nm is harmful to some biomole-
cules and should be avoided for biological applications, the S-
DMazo-modified DNA derivatives are promising candidates
for the photoregulation of bioreactions in vivo.
[12] a) T. Fujii, H. Kashida, H. Asanuma, Chem. Eur. J. 2009, 15,
10092 – 10102; b) P. C. Chen, Y. C. Chieh, J. C. Wu, J. Mol.
Struct.: Theochem 2005, 715, 183 – 189.
[13] a) H. Nishioka, X. G. Liang, H. Kashida, H. Asanuma, Chem.
Commun. 2007, 4354 – 4356; b) H. Nishioka, X. G. Liang, H.
Asanuma, Chem. Eur. J. 2010, 16, 2054 – 2062.
Received: August 28, 2011
Revised: October 31, 2011
Published online: December 15, 2011
[14] H. Asanuma, H. Hayashi, J. Zhao, X. G. Liang, A. Yamazawa, T.
Kuramochi, D. Matsunaga, Y. Aiba, H. Kashida, M. Komiyama,
Chem. Commun. 2006, 5062 – 5064.
[15] Azobenzene was photoisomerized from trans to cis in the duplex
state, although the ratio of the cis form obtained was lower
relative to the isomerization in the single-stranded state. See
Refs. [8b] and [9b].
Keywords: azo compounds · DNA structures · isomerization ·
molecular devices · photoregulation
.
[1] a) N. C. Seeman, P. S. Lukeman, Rep. Prog. Phys. 2005, 68, 237 –
270; b) M. K. Beissenhirtz, I. Willner, Org. Biomol. Chem. 2006,
4, 3392 – 3401; c) A. Condon, Nat. Rev. Genet. 2006, 7, 565 – 575;
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