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: 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¢-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. 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Liang et al. 1560 FEBS Journal 277 (2010) 1551–1561 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... al 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 Photoregulation of DNA transcription Please note: As a service to our authors and readers, this journal... online version of this article Photoregulation of DNA transcription Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be . Photoregulation of DNA transcription by using photoresponsive T7 promoters and clarification of its mechanism Xingguo Liang 1 ,. 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.