Báo cáo khoa học: Photoregulation of DNA transcription by using photoresponsive T7 promoters and clarification of its mechanism doc

Photoregulation of DNA transcription by using
photoresponsive T7 promoters and clarification of its
mechanism
Xingguo Liang
1
, Ryuji Wakuda
1
, Kenta Fujioka
1
and Hiroyuki Asanuma
1,2
1 Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Japan
2 CREST, Japan Science and Technology Agency (JST), Kawaguchi, Japan
Introduction
Recently, artificial control of gene expression has
gained attention because of its promising applications
in cell biology, pharmacology, and bionanotechnology
[1–5]. Artificial regulation of biological processes can
be used as a robust tool for investigating the mecha-
nism of particular biological phenomena in living cells
[6–8]. One of the most powerful strategies is to cova-
lently attach a photoswitch to the target biological
compound so that its corresponding biological func-
tion can be precisely triggered at an exact location and
time simply through light irradiation [9–15]. Several
photoresponsive systems using photocaged nucleic
acids, proteins or other ligands have been reported
[16–21]. Another strategy is to manipulate sensory
photoreceptors of cells that regulate plant growth and
development in response to light signals using bioengi-
Keywords

azobenzene; modified DNA; photoregulation;
T7 promoter; transcription
Correspondence
X. G. Liang, Department of Molecular
Design and Engineering, Graduate School of
Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan
Fax: +81 52 789 2528
Tel: +81 52 789 2488
E-mail:
H. Asanuma, Department of Molecular
Design and Engineering, Graduate School of
Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan
Fax: +81 52 789 2528
Tel: +81 52 789 2488
E-mail:
(Received 10 December 2009, revised 11
January 2010, accepted 15 January 2010)
doi:10.1111/j.1742-4658.2010.07583.x
With the use of photoresponsive T7 promoters tethering two 2¢-methylazo-
benzenes or 2¢,6¢-dimethylazobenzenes, highly efficient photoregulation
of DNA transcription was obtained. After UV-A light irradiation
(320–400 nm), the rate of transcription with T7 RNA polymerase and a
photoresponsive promoter involving two 2¢,6¢-dimethylazobenzenes was
10-fold faster than that after visible light irradiation (400–600 nm). By
attaching a nonmodified azobenzene and 2¢,6¢-dimethylazobenzene at the
two positions, respectively, and by utilizing the different cis fi trans
thermal stability between cis-nonmodified azobenzene and cis-2¢,6¢-dimethy-
lazobenzene, four species of T7 promoter (cis–cis, trans–cis, cis–trans, and

trans–trans) were obtained. The four species showed transcriptional activity
in the order of cis–cis > cis–trans > trans–cis > trans–trans. Kinetic
analysis revealed that the K
m
for the cis–cis promoter (both of the
introduced azobenzene derivatives were in the cis form) and T7 RNA
polymerase was 68 times lower than that for the trans–trans form,
indicating that high photoregulatory efficiency was mainly due to a
remarkable difference in affinity for RNA polymerase. The present
approach is promising for the creation of biological tools for artificially
controlling gene expression, and as a photocontrolled system for supplying
RNA fuel for RNA-powered molecular nanomachines.
Abbreviations
Azo, nonmodified azobenzene (attached to
D-threoninol via an amide bond); DM-azo, 2¢,6¢-dimethylazobenzene; M-azo, 2¢-methylazobenzene;
RNAP, RNA polymerase.
FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS 1551
neering approaches. As a universal approach, a light-
switchable promoter system that can be attached
upstream of any target gene has been constructed by
synthesizing a fusion protein consisting of a plant phy-
tochrome (tetrapyrrole chromophore) and a promoter-
binding domain [22].
For light-switching DNA functions, we have devel-
oped a photoresponsive DNA by introducing azoben-
zene moieties that can be reversibly photoisomerized
between trans and cis forms [23–25]. By photoswitching
DNA hybridization, DNA primer extension by DNA
polymerase and RNA digestion by RNase H can be
successfully photoregulated [26,27]. Asanuma et al. also

demonstrated another photoswitching strategy with
azobenzenes: photoregulation of DNA transcription
with a photoresponsive T7 promoter constructed by
attaching azobenzene moieties to the backbone via
d-threoninol linkers [25,28,29]. In this case, a partial
structural change (not the complete formation and
dissociation of DNA duplexes) caused by photoisomer-
ization of azobenzenes resulted in photoregulation
[25,28]. We found that the simultaneous introduction
of two azobenzenes into the promoter at specific
positions facilitated such photoswitching [28]. However,
clear-cut photoswitching of DNA transcription was not
realized, probably because the photoisomerization of
nonmodified azobenzene (Azo) did not cause sufficient
change in the duplex structure [28]. Additionally, the
detailed mechanism of the photoregulation was not
clarified, because we failed to obtain every species,
especially the trans–cis and cis–trans forms. For
efficient photoregulation of gene expression, the
photoregulation mechanism should be clarified and a
robust photoresponsive promoter should be developed.
In the present study, 2¢,6¢-dimethylazobenzene
(DM-azo), a more efficient photoswitch for regulating
DNA hybridization [30], was introduced into a T7
promoter instead of Azo (Fig. 1). Clear-cut on–off
photoregulation of DNA transcription was obtained
because of the efficient suppression of T7 RNA
polymerase (RNAP) binding to the DM-azo in the
trans form. By attachment of Azo at one position and
DM-azo at another position on the T7 promoter, all

four species, cis–cis, trans–cis, cis–trans, and trans–
trans, were individually obtained. The detailed mecha-
nism of photoregulation was examined by comparing
their transcriptional activities.
Results
Photoresponsive T7 promoter involving
2¢-methylazobenzene (M-azo) and DM-azo
As shown in Fig. 1, two azobenzene moieties were
additionally introduced into the nontemplate strand of
the T7 promoter at position )9 (in the RNAP recogni-
tion region) and position )3 (in the unwinding region),
respectively. This design has been previously shown to
give the highest efficiency of photoregulation when
Azos are used [28]. After transcription, a 17-nucleotide
RNA product is produced. The conversion of
transcription was measured by PAGE analysis (the
RNA product was labeled with [
32
P]ATP[aP]) and the
photoregulatory efficiency of transcription (a) was
Fig. 1. Sequences of photoresponsive T7
promoters and the structures of Azo and its
derivatives (M-azo and DM-azo) used in this
study.
Photoregulation of DNA transcription X. G. Liang et al.
1552 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS
calculated, defined as the ratio of the amount of
transcript after UV light irradiation with respect to
that after visible light irradiation.
The photoregulatory efficiency a for conventional

Azo was 4.7 under the conditions employed, indicating
that the transcription product after UV light irradia-
tion was 4.7 times greater than that after visible light
irradiation (Fig. 2). When a modified T7 promoter
involving two M-azos or DM-azos was used, the
photoregulatory efficiency was significantly improved;
a-values for M-azo and DM-azo were 6.6 and 10.1,
respectively (Fig. 2). In particular, transcription for
DM-azo was greatly suppressed after visible light irra-
diation, although the transcription efficiency decreased
to some extent after UV light irradiation as compared
with Azo and M-azo (Figs 2A and S1C). As a result,
clear-cut on–off photoregulation of transcription was
realized with DM-azo as the photoswitch. Notably,
about 40% of the transcriptional activity, as compared
with the native T7 promoter, remained after UV light
irradiation even when two DM-azos were introduced
(Fig. S1C).
In the above cases, the concentration of promoter
DNA was 2.0 lm. Highly efficient photoregulation was
also obtained at lower concentrations. For example,
a for DM-azo was as high as 11.8 at 0.2 lm (Fig. S1).
For all of these cases, as compared with native T7 pro-
moter, no severe decrease in the transcription activity
of modified promoters was observed after UV light
irradiation (the relative activity was about 30–55%).
Interestingly, when the photoresponsive T7 promoter
was attached to a green fluorescent protein gene whose
coding region is 714 bp long, a was as high as 9.6,
even for Azo at a DNA concentration of 6.7 nm

(Fig. S2) [31].
Another benefit of using DM-azo as the photoswitch
is the extremely high thermal stability of its cis
form. The cis azobenzene derivatives usually isomerize
gradually to the trans form in the dark. Low thermal
stability causes problems for clear-cut photoswitching,
especially when the sample cannot be irradiated during
the whole reaction process and the reaction time is
long. At the temperature of the transcription reaction
(37 °C), the half-life of cis-DM-azo is 14 days, which
is about eight-fold longer than that of Azo [30]. Thus,
a photoresponsive T7 promoter involving DM-azo has
promise either in vivo or in vitro for clear-cut photos-
witching at a wide range of temperatures and time
intervals.
Kinetic analysis of transcription with T7
promoters involving various isomers of DM-azo
Interestingly, transcription with the T7 promoter,
which has high sequence specificity, proceeded at a
high rate even with the insertion of two DM-azos.
Moreover, both the backbone and side chains of the
DNA duplex are changed by introducing azobenzene
on non-natural d-threoninol. In addition, transcription
was remarkably accelerated for the cis but not the
trans form, with the former reportedly causing much
more distortion of the duplex structure [24,32]. To
explain the large difference in transcription between
UV and visible light irradiation, the K
m
and k

cat
of the
corresponding species were determined through kinetic
analyses, although trans fi cis photoisomerization is
usually difficult, owing to strong stacking interactions
between trans-DM-azo and base pairs [25]. As
ATP
product
Transcription
1427635 8
Vis UVVisUV VisUV VisUV
Nat Azo M-azo DM-azo
Photoregulatory efficiency (
α
)
Nat Azo M-azo DM-azo
1
5
10
A
B
Fig. 2. Photocontrol of the transcription reaction with the T7 pro-
moter tethering various photoresponsive molecules. (A) PAGE pat-
terns of RNA products after reaction at 37 °C for 2 h after either
visible (Vis) or UV light irradiation. (B) Photoregulatory efficiency (a)
of native T7 promoter (Nat), and T7 promoter with Azo, M-azo, and
DM-azo. a is defined as the ratio of the amount of transcript after
UV light irradiation with respect to that after visible light irradiation.
X. G. Liang et al. Photoregulation of DNA transcription
FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS 1553

measured by the change in UV ⁄ visible light spectra
after UV light irradiation at 37 °C, only about 35% of
the cis form was obtained in total (data not shown).
As there were two DM-azos, the proportion of the T7
promoter in the cis–cis form, in which both DM-azos
took the cis form, was only about 12% (0.35 · 0.35)
of the total promoter content. Here, trans fi cis isom-
erization of the DM-azos did not change greatly with
the sequences of adjacent base pairs (data not shown).
For investigation of the parameters of the cis–cis form,
a 35-nucleotide nontemplate strand involving two
DM-azos was first irradiated with UV light in the sin-
gle-stranded state to facilitate trans fi cis isomeriza-
tion, and then annealed with the template strand to
form the duplex. By this approach, 65% of the total
DM-azo was isomerized to the cis form, and accord-
ingly, about 42% (0.65 · 0.65) of the total was
obtained as the cis–cis form. The other 58% consisted
of the trans–trans (  12%), trans–cis ( 23%) and
cis–trans ( 23%) forms. Here, trans–cis means that
DM-azo takes the trans form at position )9 and the
cis form at position )3 in the modified promoter. On
the other hand, cis–trans means that DM-azo takes the
cis form at position )9 and the trans form at position
)3. The terms will always be shown in this order: the
azobenzene moiety at position )9 comes first. As cis-
DM-azo is extremely thermally stable at 37 °C, the
effect of cis fi trans thermal isomerization on the reac-
tion dynamics is negligible.
Michaelis–Menten plots of the transcription rate as a

function of the concentration of the promoter were
obtained from transcription reactions with a concentra-
tion range from 20 nm to 20 lm. For the trans–trans
form (> 96%), a higher concentration of RNAP
(150 nm) was used, because the transcription rate was
very slow. For the cis–cis form ( 42%), however, a
lower concentration (20 nm) was necessary, because the
K
m
was low. From the Michaelis–Menten plots shown
in Fig. 3, the K
m
of the cis–cis form was 0.15 lm and
that of the trans–trans form was as great as 10.3 lm.
Assuming that the trans–cis and cis–trans forms have
lower transcriptional activity than the cis–cis form
(shown to be true later in this study), the actual K
m
of
the cis–cis form should be even lower. Thus, under the
conditions used, the measured K
m
of the trans–trans
form was 68 times higher than that of the cis–cis form,
indicating that the affinity of the cis–cis form for T7
RNAP is much stronger than that of the trans–trans
form. Additionally, the measured k
cat
of the cis–cis form
(3.7 min

)1
) was estimated to be two to three times larger
than that of the trans–trans form. As a result, the speci-
ficity constant (k
cat
⁄ K
m
) of the cis–cis form was about
200 times larger than that of the trans–trans form.
Obviously, the remarkable difference in the transcrip-
tion rate between UV and visible light irradiation was
mainly caused by the marked difference in K
m
. The
difference in binding affinity between the trans–trans
and cis–cis forms was also directly observed using
surface plasmon resonance analysis (Fig. S3).
Transcriptional activity of the photoresponsive
T7 promoter involving one trans-DM-azo and one
cis-Azo
As reported previously, the role of azobenzene in
photoregulation depends on its position on the
promoter [28]. When only a single Azo was introduced
0
50
100
150
0 5 10 15 20
Concentration (µM)
V (nM ATP

incorp·min
–1
0
20
40
60
80
0 0.2 0.4 0.6
V (nM ATP
incorp·min
–1
Concentration (µ
M
)
A
B
Fig. 3. Michaelis–Menten plots of the transcription reaction (ATP
incorporation rate) for (A) trans–trans and (B) cis–cis forms by T7
RNAP as a function of promoter concentration. The concentration
of RNAP was maintained at 150 n
M for the trans–trans form and
20 n
M for the cis–cis form.
Photoregulation of DNA transcription X. G. Liang et al.
1554 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS
at position )9 (the recognition region), the cis form
showed higher binding affinity for RNAP than the
trans form. At position )3 (in the unwinding region),
on the other hand, the cis form showed higher reactiv-
ity than the trans form. Similar results were obtained

when only one DM-azo was introduced; a for positions
)3 and )9 was 1.2 and 1.8, respectively (Fig. S4).
Although the difference between the trans and cis forms
was less than two-fold in both cases, simultaneous
introduction of two azobenzenes at both positions sur-
prisingly raised a to as high as 10.1 (Fig. 2B). To clarify
the mechanism of this powerful synergy, the activity
and kinetic parameters of the trans–cis and cis–trans
forms should be investigated. However, it is very diffi-
cult to separate these two species from mixtures com-
prising four isomers [28]. As demonstrated previously,
cis-DM-azo was about 10-fold more thermally stable
than cis-Azo [30]. In the present study, we used the sig-
nificant difference in thermal stability between cis-DM-
azo and cis-Azo to prepare trans–cis and cis–trans spe-
cies separately.
Our strategy is presented in Fig. 4. To obtain the
cis–trans form, for example, DM-azo was introduced
at position )9 and Azo at position )3. After UV light
irradiation in the single-stranded state, the proportions
of cis–cis, trans–cis, cis–trans and trans–trans forms
were 50%, 27%, 15%, and 8%, respectively (Table 1).
As the half-lives of cis-DM-azo and cis-Azo are
90 min and 12 min, respectively, at 90 °C, the propor-
tions of trans–cis and cis–trans species, respectively,
changed to 1% and 49.4% after incubation at 90 °C
for 1 h in the dark (Table 1). Although the proportion
of the trans–trans form increased to 48.6% after incu-
bation, its influence on the activity measurement of the
cis–trans [cis-DM-azo()9)-trans-Azo()3)] species can

be ignored, owing to its very low activity (Fig. 2). Sim-
ilarly, the trans–cis [trans-Azo()9)-cis-DM-azo()3)]
species (49.4%) could be obtained by introducing a
DM-azo at position )3 and an Azo at position )9
(Table 1). Here, we assumed that the introduction of
Azo in place of DM-azo to obtain cis–trans and trans–
cis species does not change the photoregulation mecha-
nism, although the K
m
and k
cat
of transcription might
be slightly changed. This assumption is reasonable,
because the duplex structures involving modified or
nonmodified azobenzene moieties are almost the same,
especially in the trans form (data not shown).
The results of transcription showed that the activity
of the cis–trans species was higher than that of the
trans–cis species (Table S1). At a lower promoter
concentration, the transcription rate of the cis–trans
species was about two-fold higher than that of the
trans–cis species. When the concentration was high
enough (2.0 lm), the transcription rate tended to be
the same, indicating the saturation of DNA. For all
four species, the level of activity was in the following
order: cis–cis > cis–trans > trans–cis > trans–trans.
Kinetic analyses for cis–trans [cis-DM-azo()9)-trans-
Azo()3)] and trans–cis [trans-Azo()9)-cis-DM-azo()3)]
species (Fig. 5) gave measured K
m

values of the
cis–trans and trans–cis species to be 0.24 and 1.62 lm,
respectively. Considering that the proportion was only
A
B
Fig. 4. Strategy for preparing trans–cis and cis–trans forms using
the marked difference in thermal stability of the cis form between
Azo and DM-azo. (A) Illustration of the reversible photoisomeriza-
tion of Azo and thermal isomerization of cis-Azo. (B) Quantitative
calculation of cis-form content after treatment.
Table 1. Proportions of the four promoter species (trans–trans,
trans–cis, cis–trans and cis–cis isomers) of two different azoben-
zene-modified DNAs involving one DM-azo and one Azo.
Species
Contents (%)
Under UV light
a
90 °C, 1 h
b
X
1
, Azo
X
2
, DM-azo
X
1
, DM-azo
X
2

, Azo
X
1
, Azo
X
2
, DM-azo
X
1
, DM-azo
X
2
, Azo
cis–cis 50 50 1.0 1.0
trans–cis 27 15 40 1.4
cis–trans 15 27 1.4 40
trans–trans 8 8 58 58
a
The modified DNA in the single-stranded state was irradiated
under UV light for 5 min.
b
After UV light irradiation, the samples
were incubated at 90 °C for 1 h.
X. G. Liang et al. Photoregulation of DNA transcription
FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS 1555
49.3% cis–trans or trans–cis in the corresponding solu-
tions (Table 1), the actual K
m
should be somewhat
lower. As summarized in Table 2, the order of binding

affinity (1 ⁄ K
m
) of RNAP was cis–cis > cis–trans >
trans–cis > trans–trans, which was the same as the
order of transcriptional activity. These results suggest
that the K
m
is the rate-determining factor for transcrip-
tion by the photoresponsive promoter. The fact that
the cis–cis and cis–trans species had similar K
m
values,
and that the cis–cis and trans–cis species showed
marked differences in the K
m
, indicated that the
trans fi cis isomerization of the photoswitch at posi-
tion )9 caused a great change in binding affinity.
Additionally, the k
cat
of the cis-cis species was five
times higher than that of the cis–trans species, indicat-
ing that the cis isomer at position )3 was favorable for
the transcription reaction (Table 2). This was also true
when the azobenzene moiety at position )9 was in the
trans form; the trans–cis species showed a higher k
cat
than the trans–trans species. Thus, trans fi cis isomeri-
zation of the photoswitch at position )3 mainly influ-
enced the catalytic activity. Taking these findings

together, the significant synergistic effect of introduc-
ing two azobenzene moieties can be explained as fol-
lows: only when both azobenzene moieties are in the
cis form are both lower K
m
and higher k
cat
attained
simultaneously.
Discussion
With the use of azobenzenes at the two ortho positions
modified with methyl groups (DM-azo), clear-cut pho-
toregulatory transcription was achieved. Although the
transcriptional activity was reduced to some extent as
compared with the native system, owing to chemical
modification, the activity of the cis–cis form was fairly
acceptable. This result seems to be in conflict with our
previous results showing that nonplanar cis-azobenzene
destabilizes the DNA duplex structure by causing a
larger structural change, owing to its causing more sig-
nificant steric hindrance than the trans form [24,25].
However, this discrepancy can be adequately explained
by comparing our results with the crystal structure of
the T7 RNAP–promoter complex analyzed by other
groups. Cheetham et al. reported that the specificity
loop of T7 RNAP binds to the major groove of the
promoter from position )8 to position )11 through
sequence-specific hydrogen-bonding interactions
between protein side chains and base pairs [33,34]. At
the same time, the specificity loop also binds to the

melted template strand at the TATA box region ()1to
)4) (Fig. S5) [35,36].
Figure 6 shows molecular modeling structures of the
photoresponsive T7 promoter involving two DM-azos
in the absence of T7 RNAP. For the trans–trans form,
0
5
10
15
20
0 0.5 1 1.5 2 2.5
V (nM ATP incorp·min
–1
)
Concentration (µM)
Concentration (µ
M)
0
5
10
15
20
0 0.5 1 1.5 2 2.5
V (nM ATP incorp·min
–1
)
A
B
Fig. 5. Michaelis–Menten plots of the transcription reaction (ATP
incorporation rate) for (A) trans–cis and (B) cis–trans forms as a

function of promoter concentration. The two forms were prepared
as shown in Fig. 4. The concentration of RNAP was maintained at
20 n
M.
Table 2. Kinetic parameters for the four promoter species deter-
mined from Michaelis–Menten plots. The data for the cis–cis and
trans–trans forms were obtained when two DM-azos were intro-
duced into the T7 promoter at positions )9 and )3.
Species K
m
(10
)6
M) K
cat
(min
)1
)
K
cat
⁄ K
m
(10
6
M
)1
Æmin
)1
)
cis–cis 0.15 3.7 24
trans–cis 1.5 1.6 1.1

cis–trans 0.24 0.78 3.3
trans–trans 10.3 1.3 0.12
Photoregulation of DNA transcription X. G. Liang et al.
1556 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS
two trans-DM-azos intercalated and stacked strongly
with adjacent base pairs. Obviously, the presence of
two trans-DM-azos causes a significant change in both
the groove structure and length of the promoter
duplex, restricting the binding of T7 RNAP (Fig. 6A).
That is, a large amount of energy is required for
RNAP to flip out both intercalated DM-azos, allowing
binding at the major groove. At position )9, the
hydrogen bonds between T7 RNAP and the promoter
appear to be disrupted; at position )3, the intercalated
trans-DM-azo hinders melting of the TATA box. The
introduction of trans-DM-azo, rather than trans-Azo,
has shown stabilizing effects on the duplex structure
[30]. Accordingly, the effect of suppressing RNAP
activity was enhanced using DM-azo involving two
ortho-methyl groups instead of Azo (Fig. 2).
In the case of the cis–cis form, as shown in Fig. 6B,
two cis-DM-azos tend to flip out to the minor groove.
For both position )9 and position )3, the base pairs
adjacent to each DM-azo become close to each other
at the major groove. As a result, the cis–cis promoter
can assume a conformation at the major groove that is
similar to the native one, allowing RNAP to bind
easily. Furthermore, the cis-DM-azo moiety is easily
pushed out from the minor groove owing to RNAP
binding, because cis-DM-azo greatly destabilizes the

duplex and the acyclic d-threoninol linker shows rela-
tively high flexibility. For the same reason, the TATA
BA
CD
–4A
–3T
–5C
–7G
–8A
–9G
–10T
–2A
–8T
Major
groove
–3A
–7G
–9C
–11C
–8 A
Major
groove
–4A
–2T
–2A
–3T
–6T
–4T
–4A
–3T

–5C
–7G
–8A
–10T
–2A
–8T
Major
groove
Major
groove
–9C
–3A
–7G
–9C
–10A
–8 A
–4A
–2T
–2A
–3T
–6T
–4T
–9G
Fig. 6. Molecular modeling structures of
modified T7 promoters for (A) trans–trans
(trans-DM-azo, trans-DM-azo), (B) cis–cis
(cis-DM-azo, cis-DM-azo), (C) cis–trans
(cis-DM-azo, trans-Azo) and (D) trans–cis
(trans-Azo, cis-DM-azo) forms. Azo and ⁄ or
DM-azo were attached to the nontemplate

strand. T7 RNAP usually interacts directly
with bases )7G, )8A and )9G on the
template strand from the major groove [33].
X. G. Liang et al. Photoregulation of DNA transcription
FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS 1557
box melts much more easily, owing to the cis-DM-azo
at position )3. Because RNAP only binds to the tem-
plate strand at this region, the structure of the complex
of RNAP and the photoresponsive promoter should be
similar once the TATA box is melted. We have even
found that when only one cis-Azo is present in the
promoter at this position, the activity of the RNAP
reaction becomes slightly higher than that of the native
promoter [28]. Furthermore, Martin et al. reported
that deletion of the downstream part of a nontemplate
strand after position +1 (the nontemplate strand con-
sists of only 17 nucleotides, )17 to )1) causes a two-
fold increase in k
cat
, because the TATA box becomes
easier to melt [37]. Thus, the influence of nonplanar
cis-DM-azos on transcription was greatly alleviated, so
that the cis–cis form showed a low K
m
and a large
k
cat
.
Besides the stabilization effect of trans-DM-azo at
the TATA box, the conformational change caused by

the introduction of two trans–trans DM-azos may
contribute to a lower k
cat
. First, the presence of
trans-DM-azos may suppress the incorporation of
NTPs on the template strand. Second, the synthesized
short, abortive RNA (three to eight nucleotides)
might be more difficult to elongate, owing to the
presence of trans-DM-azo. Usually, the intercalation
of trans-azobenzene in the DNA duplex causes
unwinding of the duplex to some extent [31]. A similar
situation might occur for the cis–trans form, which
has a lower k
cat
even when the azobenzene moiety at
position )3 takes the trans form (Fig. 6D).
As shown in Fig. 2, the transcription rate under UV
light irradiation increased by about 10-fold as com-
pared with that after visible light irradiation, even
though only about 10% of the total T7 promoters
took the cis–cis form after UV light irradiation under
the present transcription conditions. Because k
cat
⁄ K
m
of the cis–cis form was found to be about 200-fold lar-
ger than that of the trans–trans form (Table 2), 10%
of the cis–cis form results in 10-fold more efficient
transcription, especially at a lower DNA concentra-
tion. The kinetic analysis also showed that the con-

structed photoresponsive T7 promoter has an
extremely high potential for photoswitching transcrip-
tion if the efficiency of photoisomerization can be
improved.
Another interesting point is that the yield of tran-
script with modified T7 promoter after UV light irradi-
ation did not decrease abruptly as compared with that
of the native promoter, although the proportion of the
cis–cis form was only about 10%. One possible reason
is that the concentration of promoters used here is
much higher than that of T7 RNAP. Accordingly, the
concentration of modified promoters in the cis–cis
form may be enough to be used by RNAP for tran-
scription, especially when the K
m
is smaller. All of the
results indicate that the modified promoter in the cis–
cis form should have similar activity as the native one
[28].
In conclusion, by introducing two DM-azos at posi-
tions )9 and )3 of the T7 promoter, clear-cut photore-
gulation of DNA transcription was obtained. Both
sufficient suppression of transcription after visible light
irradiation (trans–trans form) and a limited decrease in
activity after UV light irradiation (cis–cis form) con-
tributed to efficient photoregulation. Kinetically, the
marked difference in binding affinity for RNAP (K
m
)
between the trans–trans and cis–cis forms strongly sup-

ports such clear-cut photoregulation. By photoswitch-
ing gene expression simply with light irradiation,
photoresponsive promoters can be powerful tools for
clarifying the mechanisms of bioreactions or for appli-
cations in genetic therapy. Furthermore, on–off photo-
regulation of DNA transcription is promising as a
photoswitched supplier of RNA fuel for driving
nanodevices [5].
Experimental procedures
Materials
Oligonucleotides consisting of only native bases were sup-
plied by Integrated DNA Technologies, Inc. (Coralville, IA,
USA). Oligonucleotides involving azobenzene residues were
synthesized on an ABI 394 Nucleic Acid synthesizer
(Applied Biosystems, Foster City, CA, USA). Purification
was performed by either PAGE or RP-HPLC with a
LiChrospher 100 RP-18(e) column (Merck, Darmstadt,
Germany). The corresponding phosphoramidite monomers
were synthesized according to a protocol reported previ-
ously [24,25,30]. Concentrations of all oligonucleotides were
determined by UV ⁄ visible spectroscopy analysis within
10% error. The molecular extinction coefficients (e) of Azo
and DM-azo residues are 7.0 · 10
3
molÆL
)1
Æcm
)1
and
2.1 · 10

4
molÆL
)1
Æcm
)1
, respectively. All modified oligonu-
cleotides were characterized by MALDI-TOF MS (Auto-
FLEX mass spectrometer in positive ion mode, Bruker). T7
RNAP was purchased from Takara Bio Inc. (Shiga, Japan).
The concentration of T7 RNAP was determined by the
absorbance at 280 nm with an extinction coefficient of
1.4 · 10
5
m
)1
cm
)1
[28,38].
Transcription reaction catalyzed by T7 RNAP
Typical transcription was carried out as follows. To a
20 lL solution containing duplexes of the T7 promoter,
0.5 mm each NTP, 2 lCi of [
32
P]ATP[aP], corresponding
Photoregulation of DNA transcription X. G. Liang et al.
1558 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS
buffer [final concentration: 40 mm Tris ⁄ HCl buffer
(pH 8.0), 2 mm spermidine, 5 mm dithiothreitol, 24 mm
MgCl
2

, and 2 mm NaCl] and 50 U of T7 RNAP were
added, and the reaction was incubated at 37 °C for 2 h.
Then, 5 lL of reaction solution was quenched by adding
5 lL of loading buffer containing 80% formamide, 50 mm
EDTA, and 0.025% bromophenol blue. The mixture was
subjected to electrophoresis on a 20% denaturing polyacryl-
amide gel containing 7 m urea. After exposure to an imag-
ing plate (BAS-MS2340; Fujifilm, Tokyo, Japan),
radioisotopic images were analyzed with an FLA-3000 bio-
imaging analyzer (Fujifilm).
To obtain data for the Michaelis–Menten plots, tran-
scription was performed by changing the concentration of
the promoters. Except for the cis–cis form, for which
20 nm RNAP was used. Other conditions were the same as
described above. The yield of 17-nucleotide RNA was
maintained below 5%. Each experiment was performed at
least twice. The K
m
was calculated using kaleida-
graph 3.5J (Synergy Software). The k
cat
was calculated
with the formula k
cat
= V
max
⁄ [RNAP].
Photoisomerization of azobenzene derivatives on
photoresponsive T7 promoters
For cis fi trans photoisomerization, a solution containing

promoter duplexes was irradiated at 37 °C for 1 min with
visible light (400–600 nm) from a Xenon lamp (Hamamatsu
Photonics, Shizuoka, Japan) through an L-42 filter (Asahi
Technoglass Cooperation, Chiba, Japan). For photoswitch-
ing experiments, trans fi cis photoisomerization was
achieved by irradiating the promoter duplex at 37 °C with
a UV-A (320–400 nm) fluorescent lamp (FL6BL-A; Toshi-
ba Cooperation, Tokyo, Japan) for at least 5 min. To
achieve more cis isomers during kinetic analysis, trans fi
cis isomerization was performed by irradiating photore-
sponsive ssDNA at 60 ° C for 3 min with a 150 mW Xenon
lamp through a UVD-36C filter (320–400 nm). Then,
photoresponsive DNA rich in the cis form was mixed with
the template strand and incubated at 37 °C for 30 min in
the dark.
To achieve cis–trans and trans–cis isomers, a solution
containing ssDNA involving one Azo and one DM-azo was
maintained at 90 °C for 1 h after trans fi cis isomerization
as described above. Then, the template strand was added
and annealed at 37 °C for 30 min. Thereafter, solutions
involving cis isomers were maintained under dark or low-
light conditions.
Molecular modeling
The insight ii ⁄ discover 98.0 program package (Accelrys
Software Inc., San Diego, CA, USA) was used for molecu-
lar modeling to obtain energy-minimized structures by min-
imization of the conformational energy. The effects of
water and counterions were simulated by a sigmoidal, dis-
tance-dependent, dielectric function. The B-type duplex was
used as the initial structure, and amber force fields were

used for the calculation. Structures of azobenzene residues
were built using the attached graphical program.
Acknowledgements
This work was supported, in part, by a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan
(No. 21241031), by Core Research for Evolution
Science and Technology (CREST), Japan Science and
Technology Agency, Japan, and by a Grant-in-Aid for
Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan
(Nos. 20750132 and 18001001).
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Supporting information
The following supplementary material is available:
Fig. S1. Photoregulation efficiency at various

concentrations.
Fig. S2. Photoregulation efficiency for transcription of
the GFP gene initiated by the photoresponsive T7 pro-
moter involving two Azos.
Fig. S3. Direct observation of differences in binding
affinity between the trans–trans and cis–cis forms using
surface plasmon resonance analysis.
Fig. S4. Photoregulation efficiency for promoters
involving only one DM-azo.
Photoregulation of DNA transcription X. G. Liang et al.
1560 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS
Fig. S5. Illustration of the positions of azobenzene
moieties in the 3D structure of T7 RNA polymerase
binding to a native T7 promoter.
Table S1. Transcriptional activity of the four promoter
species at various concentrations.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
X. G. Liang et al. Photoregulation of DNA transcription
FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS 1561