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

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 t

photoresponsive T7 promoters and clarification of its

mechanism

Xingguo Liang1, Ryuji Wakuda1, Kenta Fujioka1and Hiroyuki Asanuma1,2

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 locafunc-tion 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: liang@mol.nagoya-u.ac.jp

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: asanuma@mol.nagoya-u.ac.jp

(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¢-dimethylazo2¢-methylazo-benzenes, 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 cisfi 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 Km 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.

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 posirecogni-tion)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 [32P]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.

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 (37C), 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 Kmand kcatof the corresponding species were determined through kinetic analyses, although transfi cis photoisomerization is usually difficult, owing to strong stacking interactions between trans-DM-azo and base pairs [25] As

ATP

product Transcription

1 2 3 4 5 6 7 8

Vis UV Vis UV Vis UV Vis UV

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.

measured by the change in UV⁄ visible light spectra

after UV light irradiation at 37C, 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, transfi 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 transfi 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

cisform 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 37C, the

effect of cisfi 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

Kmwas low From the Michaelis–Menten plots shown

in Fig 3, the Km 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 Kmof

the cis–cis form should be even lower Thus, under the

conditions used, the measured Km 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 kcatof 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 (kcat⁄ Km) 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 irradiatranscrip-tion was mainly caused by the marked difference in Km 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 20 40 60 80

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.

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 90C, the propor-tions of trans–cis and cis–trans species, respectively, changed to 1% and 49.4% after incubation at 90C 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– cisspecies does not change the photoregulation mecha-nism, although the Km and kcatof 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 Km 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 isomerizaphotoisomeriza-tion 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 lighta 90 C, 1 h b

X 1 , Azo

X2, DM-azo

X 1 , DM-azo

X2, Azo

X 1 , Azo

X2, DM-azo

X 1 , DM-azo

X2, Azo

a The modified DNA in the single-stranded state was irradiated under UV light for 5 min. bAfter UV light irradiation, the samples were incubated at 90 C for 1 h.

49.3% cis–trans or trans–cis in the corresponding

solu-tions (Table 1), the actual Km should be somewhat

lower As summarized in Table 2, the order of binding

affinity (1⁄ Km) 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 Kmis the rate-determining factor for

transcrip-tion by the photoresponsive promoter The fact that

the cis–cis and cis–trans species had similar Kmvalues,

and that the cis–cis and trans–cis species showed

marked differences in the Km, indicated that the

transfi cis isomerization of the photoswitch at

posi-tion )9 caused a great change in binding affinity

Additionally, the kcat 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 kcat than the trans–trans species Thus, transfi 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 Km and higher kcat 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 ()1 to )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

Concentration (µ M )

Concentration (µ M ) 0

5

10

15

20

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)6M ) K cat (min)1)

Kcat⁄ K m (106M )1Æmin)1)

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

B A

–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].

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 kcat, 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 Km and a large

kcat

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 kcat 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 kcat 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 kcat⁄ Km

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 photoisomerizatranscrip-tion 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–cisform 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 Kmis 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 (Km) 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

10% error The molecular extinction coefficients (e) of Azo

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

Transcription reaction catalyzed by T7 RNAP Typical transcription was carried out as follows To a

20 lL solution containing duplexes of the T7 promoter,

buffer [final concentration: 40 mm Tris⁄ HCl buffer

(pH 8.0), 2 mm spermidine, 5 mm dithiothreitol, 24 mm

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

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

Photoisomerization of azobenzene derivatives on

photoresponsive T7 promoters

visible light (400–600 nm) from a Xenon lamp (Hamamatsu

Photonics, Shizuoka, Japan) through an L-42 filter (Asahi

Technoglass Cooperation, Chiba, Japan) For

a UV-A (320–400 nm) fluorescent lamp (FL6BL-A;

Toshi-ba Cooperation, Tokyo, Japan) for at least 5 min To

lamp through a UVD-36C filter (320–400 nm) Then,

photoresponsive DNA rich in the cis form was mixed with

the dark

To achieve cis–trans and trans–cis isomers, a solution

containing ssDNA involving one Azo and one DM-azo was

as described above Then, the template strand was added

involving cis isomers were maintained under dark or

low-light conditions

Molecular modeling

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