A thermostable azo-linker for reversible photoregulation of DNA replication

Article abstract of DOI:10.1016/j.tetlet.2008.06.027

Small molecules such as azobenzenes, one of the best reversible photo-switches, can be covalently incorporated into DNA to regulate its structures and functions with irradiation of the specific wavelengths. Using this strategy, a thermostable azobenzene l

Full text of DOI:10.1016/j.tetlet.2008.06.027

Tetrahedron Letters 49 (2008) 5087–5089
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A thermostable azo-linker for reversible photoregulation of DNA replication
Qi Wang, Long Yi, Liangliang Liu, Chuanzheng Zhou, Zhen Xi *
State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecules such as azobenzenes, one of the best reversible photo-switches, can be covalently incor-
porated into DNA to regulate its structures and functions with irradiation of the specific wavelengths.
Using this strategy, a thermostable azobenzene linker was employed to construct modified oligodeoxy-
nucleotides, and we successfully achieve reversible photoregulation of DNA replication in vitro with short
irradiation time. Five minutes UV irradiation for regulating trans?cis transformation can minimize DNA
damage, still ensure the polymerase reaction of cis-form. Formation of DNA hairpin structure can also be
controlled by photoregulation using this linker.
Received 9 April 2008
Revised 22 May 2008
Accepted 6 June 2008
Available online 12 June 2008
Keywords:
DNA replication
Reversible photoregulation
Thermostable azo-linker
Hairpin DNA
Ó 2008 Elsevier Ltd. All rights reserved.
Much research has been centered on modified oligonucleotides,
and various types of biological and chemical functionalization have
recently been achieved. For example, small molecules such as azo-
reactions in vitro. Considering the recognizability of KFexo- poly-
merase, we design and synthesize DNA 5–7 including an azobenz-
6
1
ene linker for photoinducing cisMtrans isomeriaztion. When the
DNA synthesis with KFexo- polymerase was achieved at pH 7.9
and 37 °C in the dark in the presence of oligomer 5, 5.4% 28-mer
DNA was produced, while the same reaction performed under UV
irradiation (347 ± 40 nm), nearly no DNA elongation was found. It
benzenes, one of the best reversible photo-switches, can be cova-
lently incorporated into DNA to regulate its conformation and
2
properties with irradiation of the specific wavelengths. One of
the problems caused by the modification of reported azobenzene
is degradation of cis-azobenzene, which is normally not thermosta-
3
ble enough at 37 °C with T1/2 less than 10 h. In fact, introducing an
inductive electron-withdrawing group to azobenzene’s meta-posi-
4
ton, the obtained compound can be an excellent photoswitch.
0
Herein we choose 3,3 -azo-di-benzyl alcohol (2) as a photoregula-
tor for two reasons: (I) it can connect two DNA sequences as a
linker rather than a side chain fragment, this linker may give
attractive properties because it would be in a different proximity
imposed by the photoisomerization; (II) it exhibits thermostability
for practical use in many biological studies at physiological
conditions.
Compound 2 was easily obtained from 3-nitrophenylmethanol,5
and its photoisomerization efficiency and thermostability are
4
excellent. Compound 2 can be introduced into backbone of DNA
using conventional phosphoramidite chemistry (Scheme 1). These
modified oligodeoxynucleotides 5–7 contain five, six, and seven
0
bases, respectively, at 3 terminus following the linker, which can
form base pairs with those bases at the other side of the linker.
Compound 2 is an excellent candidate as a photoswitch with
good thermal stability of the cis-form.4 The modified oligodeoxy-
nucleotides containing 2 as a linker were used in DNA polymerase
Scheme 1. The synthetic scheme of modified azobenzene and the DNA sequences
used in this study; L indicates the azobenzene linker derived from 4. Reagents and
conditions: (i) SnCl
DMAP, CH Cl ; (iii) diisopropylamine, 1H-tetrazole, CH
tetraisopropylphosphorodiamidite, two steps yield, 60%.
2 2 3 2
, NaOH, H O/CH CH OH, reflux, 85%; (ii) DMT-Cl, pyridine,
0 0
2 2
Cl , 2-cyanoethyl-N,N,N ,N -
*
Corresponding author. Tel./fax: +86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
2
2
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.06.027

5
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Q. Wang et al. / Tetrahedron Letters 49 (2008) 5087–5089
should be noted that each linker in the present study nearly equals
to 2 nt, and mobility of 28-mer DNA was observed from the gel
(Fig. 1, lane 3). The UV irradiation causes no significant effects on
the enzymatic activity, as confirmed by careful control experi-
ments. At the same reaction condition, the yields of 30-mer DNA
from oligomer 6 with and without UV irradiation are 26.3% and
4
8.0%, respectively, which imply that UV regulation has significant
effect on the reaction in oligomer 6. It is interesting to note that the
UV irradiation causes no significant difference on the reaction of
oligomer 7, as confirmed by the fact that the reaction with and
without UV irradiation has 27.5% and 28.6% yields of 32-mer
DNA, respectively. These experiments were repeated three times
to lead nearly similar results, and the photoregulation of DNA rep-
lication depends on the DNA lengths following azobenzene linker
0
at 3 terminus, namely, high selectivity but low yield for oligomer
5, both selectivity and high yield for oligomer 6, and no selectivity
for oligomer 7.
Scheme 2. A possible mechanism for DNA polymerase reactions with different
lengths of DNA; DNA elongation part, red line; reaction template, black line; short
primer, green and purple lines; long primer, blue and cyan lines; base pairs exist
between green and purples lines or blue and cyan lines.
These interesting experiment results imply a possible reaction
mechanism as shown in Scheme 2. Considering both the configura-
tion of azobenzene and DNA sequences, DNA chains could form du-
plex intramolecularly or/and intermolecularly. As a result, the DNA
0
sequences at 3 terminus of one chain can act as a primer and an-
form is nearly two times (1.83:1) higher than that for the cis-form.
We suppose that if one polymerase attaches one reaction site of the
trans-form, it can very quickly transfer to another reaction site of
this DNA by a ‘supra-intramolecular jumping’ rather than the nor-
mal diffusion from polymerase to DNA, which leads to expedite the
reaction in this case.
other chain as a template. While DNA with the cis-form linker can
form hairpin structure, the oligonucletides provide both template
and primer by itself for DNA replication.
7
Short base-pairing length of oligodeoxynucleotide (such as in
oligomer 5), giving relatively weak interaction for base-pairing,
leads to either the low yield by weak binding from polymerase in
dark, or no yield as the inefficacy of polymerase binding to the very
short stemmed hairpin structure in the cis-form azobenzene linker
under UV for oligomer 5.
Considering the base-pair numbers and the experimental
results, DNA may prefer duplex for the trans-form. While for the
cis-form, the hairpin structure in this case can provide both primer
and template for DNA polymerase. As a result, both DNA syntheses
in the dark and under UV irradiation were observed in oligomer 6.
Note that two DNA with duplex can provide two reaction sites,
while there is only one in the hairpin structure. Because the total
reaction sites of both cases in the present reaction conditions are
the same, the polymerases will diffuse to DNA to give the similar
reaction velocity. However, the DNA synthesis yield for the trans-
Increasing the DNA length as in oligomer 7 gives a better
hairpin structure for both cis- and trans-azobenzene linker, due
to entropy effect of intramolecular base-pairing interactions that
overcome the stereo effect of azobenzene even in the trans-form.
So the DNA synthesis yields with and without UV irradiation are
similar in oligomer 7, which is also the case as in the polymerase
reaction of TATCAGACATATTAATLATTAATAT (data not show).
Oligomer 6 was further chosen for the investigation of rever-
sible photoregulation (Fig. 2). The DNA syntheses in the dark and
under UV irradiation for 1 h give similar results as that found in
Figure 1, and the yields ratio is nearly 2:1. Half an hour reaction
under UV irradiation followed by half an hour reaction under visi-
ble light gave 27.1% yield. One hour reaction under UV irradiation
gave 16.2% yield, while that in dark 31.3%. These results imply that
after half an hour UV irradiation, the followed visible light irradia-
tion makes cis?trans transformation of the linker that causes
opening of the hairpin structure (Scheme 3).
In practical application of photoregulation, the UV irradiation
time for artificial regulation on DNA replication should be as short
as possible to avoid possible DNA damage. In this work, a thermo-
stable azobenzene linker was designed to overcome this shortcom-
Figure 1. Polyacrylamide gel electrophoresis patterns for Klenow fragment of
Escherichia coli DNA polymerase I (KFexo-, 0.02 U/
lL) reactions at pH 7.9, 37 °C for
0
3
2
3
2
3
0 min. These oligodeoxynucleotides (30 nM) were 5 - P-end-labled using [c- P]
ATP and T4 polyynucleotide kinase. All oligodeoxynucleotides were irradiated with
light over 540 nm for 10 min before DNA polymerase reaction to make sure that
azobenzene in the oligodeoxynucleotides is of trans-form. The oligodeoxynucleo-
tides used in the reaction under UV irradiation were firstly irradiated by UV light for
5
min, and then added to the reaction tube. Lanes 1 and 11: markers (18-mer, 30-
mer, and 38-mer); S1–S3 for 5–7, respectively; lanes 2, 5, and 8: control
experiments, reaction without KFexo-; lanes 3, 6, and 9: with modified oligode-
oxynucleotides in the dark; lanes 4, 7, and 10: with modified oligodeoxynucleotides
under UV irradiation. DNA syntheses were quantitated using Typhoon TRIO
Phosphorimager.
Figure 2. The reaction yields of 6 irradiated by a different light (see Fig. S3 for the
gel patterns).

Q. Wang et al. / Tetrahedron Letters 49 (2008) 5087–5089
5089
extra fast moving bands were slightly visible after 1 h UV irradia-
tion, indicating possible DNA radiation damage for long time UV
irradiation (Fig. 3, lane 5). Five minutes UV irradiation can mini-
mize DNA damage, still ensuring the polymerase reaction of cis-
form.
In conclusion, the results reported here indicate that reversible
photoregulation for the DNA replication can be achieved through
incorporating a thermostable azobenzene linker. Formation of
DNA hairpin structure can also be controlled by photoregulation
using this linker. We are exploring the potential applications of this
thermostable linker to regulate different DNA/RNA functions.
Acknowledgments
This work was supported by the National Key Project for
Basic Research of China (2003CB114403), National Natural Science
Foundation of China (20272029, 20572053, 20421202, 20432010),
Ministry of Education of China (104189, B06005) and Nankai
University ISC.
Scheme 3. Reversible photoregulation of DNA hairpin and duplex structure by
UV/visible light; blue, T; red, A.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2008.06.027.
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Figure 3. Polyacrylamide gel electrophoresis patterns for DNA reactions of 6 with
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