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We have previously shown that the upper critical solution temperature-type thermoresponsive ureido polymers such as polyallylurea and poly(2-ureidoethylmethacrylate) derivatives show liquid-liquid phase separation (LLPS), also known as simple coacervation, under physiological conditions below their phase-separation temperatures ( T p ). The addition of the polymer-rich coacervate droplets that result from LLPS to a monolayer cell culture induced aggregation of cells into multicellular spheroids. In this study, we prepared a ureido copolymer, poly(vinylamine- co -vinylurea), with azobenzene substituents (Azo-PVU) and demonstrated light-guided assembly and disassembly of LLPS coacervates. Azo-PVUs with T p values ranging from 10 to 52 °C were prepared by changing the azobenzene content. Ultraviolet light caused a decrease in the T p of Azo-PVU because of trans -to- cis photoisomerization of the azobenzene and irradiation with visible light increased the T p . Thus, LLPS of Azo-PVU was reversibly controlled. The coacervate droplets deposited on a dish surface were immediately dissolved by targeted UV irradiation (owing to a decrease in the T p ). Spatially controlled recruitment of proteins on the dish surface was achieved when protein solution was added to the light-patterned surface. Furthermore, the light-guided deposition of coacervates resulted in the spatiotemporal transformation of monolayer cells to aggregates. This light-controlled LLPS will allow the preparation of novel liquid-based materials for biomolecular and cellular engineering.

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials, and nanofactories. Present here is an entirely light-induced DNA walker based on orthogonal photocontrol. Implementing two azobenzene derivatives, S-DM-Azo and DM-Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non-autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back-and-forth directions as desired, and is regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non-autonomous progressive molecular motors, ultimately facilitating their application at the nanoscale., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 10-23 DNAzyme is an artificially developed functional oligonucleotide that can cleave RNA in a sequence-specific manner. In this study, we designed a new photo-driven DNAzyme incorporating a photoresponsive DNA overhang complementary to the catalytic core region. The photoresponsive overhang region of the DNAzyme included either azobenzene components (Azos) or 2,6-dimethyl-4-(methylthio)azobenzene units (SDM-Azos) each attached to a d-threoninol linker. When the Azos or SDM-Azos were in the trans form, the photoresponsive DNA overhang hybridized with the DNAzyme, and the RNA cleavage activity was suppressed. cis Isomerization of Azos or SDM-Azos, induced by 365 or 400 nm light, respectively, destabilized the duplex between the photoresponsive overhang and the catalytic core, and the DNAzyme recovered RNA cleavage activity. Reversible photoswitching of the DNAzyme activity was achieved by use of specific light irradiation. Further, light-dependent photoswitching of protein expression in the presence of the DNAzyme was demonstrated. Thus, this photo-driven DNAzyme has potential for application as a photocontrolled gene silencing system and a photoactivatable gene expression system., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study we constructed spherical photo-responsive microcapsules composed of three photo-switchable DNA strands. These strands first formed a three-way junction (TWJ) motif that further self-assembled to form microspheres through hybridization of the sticky-end regions of each branch. To serve as the photo-switch, multiple unmodified azobenzene (Azo) or 2,6-dimethyl-4-(methylthio)azobenzene (SDM-Azo) were introduced into the sticky-end regions via a d-threoninol linker. The DNA capsule structure deformed upon trans-to-cis isomerization of Azo or SDM-Azo induced by specific light irradiation. In addition, photo-triggered release of encapsulated small molecules from the DNA microcapsule was successfully achieved. Moreover, we demonstrated that photo-triggered release of doxorubicin caused cytotoxicity to cultured cells. This biocompatible photo-responsive microcapsule has potential application as a photo-controlled drug-release system., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photodriven DNA strand displacement by using a 2',6'-dimethylazobenzene-tethered strand and poly(l-lysine)-graft-dextran (PLL-g-Dex) as a chaperone is reported. Rapid strand displacement was reversibly induced by UV and visible-light irradiation without any toehold portion. To further improve the method, the concentration of PLL-g-Dex and the number of equivalents of the photoresponsive strand were optimised. Optimally, 64 % strand displacement was reversibly induced by alternating UV and visible-light irradiation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we report the reversible light-regulated destabilization of DNA duplexes by using azobenzene C-nucleoside photoswitches. The incorporation of two different azobenzene residues into DNA and their photoswitching properties are described. These new residues demonstrate a photoinduced destabilization effect comparable to the widely applied D-threoninol-linked azobenzene switch, which is currently the benchmark. The photoswitches presented herein show excellent photoswitching efficiencies in DNA duplexes - even at room temperature - which are superior to commonly used azobenzene-based nucleic acid photoswitches. In addition, these photoswitching residues exhibit high thermal stability and excellent fatigue resistance, thus rendering them one of the most efficient candidates for the regulation of duplex stability with light., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4'-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4'-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ < 370 nm), and 4-methylthioazobenzene-4'-carboxylic acid (S-Azo) and 2,6-dimethyl-4-(methylthio)azobenzene-4'-carobxylic acid (S-DM-Azo), which were cis-isomerized by irradiation with 400 nm visible light. Expression of green fluorescent protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340-360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage.

Photoregulation of RNA remains a challenging task as the introduction of a photoswitch entails changes in the shape and the stability of the duplex that strongly depend on the chosen linker strategy. Herein, the influence of a novel nucleosidic linker moiety on the photoregulation efficiency of azobenzene is investigated. To this purpose, two azobenzene C-nucleosides were stereoselectively synthesized, characterized, and incorporated into RNA oligonucleotides. Spectroscopic characterization revealed a reversible and fast switching process, even at 20 °C, and a high thermal stability of the respective cis isomers. The photoregulation efficiency of RNA duplexes upon trans-to-cis isomerization was investigated by using melting point studies and compared with the known D-threoninol-based azobenzene system, revealing a photoswitching amplitude of the new residues exceeding 90 % even at room temperature. Structural changes in the duplexes upon photoisomerization were investigated by using MM/MD calculations. The excellent photoswitching performance at room temperature and the high thermal stability make these new azobenzene residues promising candidates for in-vivo and nanoarchitecture photoregulation applications of RNA., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CONSPECTUS: DNA is regarded as an excellent nanomaterial due to its supramolecular property of duplex formation through A-T and G-C complementary pairs. By simply designing sequences, we can create any desired 2D or 3D nanoarchitecture with DNA. Based on these nanoarchitectures, motional DNA-based nanomachines have also been developed. Most of the nanomachines require molecular fuels to drive them. Typically, a toehold exchange reaction is applied with a complementary DNA strand as a fuel. However, repetitive operation of the machines accumulates waste DNA duplexes in the solution that gradually deteriorate the motional efficiency. Hence, we are facing an "environmental problem" even in the nanoworld. One of the direct solutions to this problem is to use clean energy, such as light. Since light does not contaminate the reaction system, a DNA nanomachine run by a photon engine can overcome the drawback of waste that is a problem with molecular-fueled engines. There are several photoresponsive molecules that convert light energy to mechanical motion through the change of geometry of the molecules; these include spiropyran, diarylethene, stilbene, and azobenzene. Although each molecule has both advantages and drawbacks, azobenzene derivatives are widely used as "molecular photon engines". In this Account, we review light-driven DNA nanomachines mainly focusing on the photoresponsive DNAs that we have developed for the past decade. The basis of our method is installation of an azobenzene into a DNA sequence through a d-threoninol scaffold. Reversible hybridization of the DNA duplex, triggered by trans-cis isomerization of azobenzene in the DNA sequences by irradiation with light, induces mechanical motion of the DNA nanomachine. Moreover we have successfully developed azobenzene derivatives that improve its photoisomerizaition properties. Use of these derivatives and techniques have allowed us to design various DNA machines that demonstrate sophisticated motion in response to lights of different wavelengths without a drop in photoregulatory efficiency. In this Account, we emphasize the advantages of our methods including (1) ease of preparation, (2) comprehensive sequence design of azobenzene-tethered DNA, (3) efficient photoisomerization, and (4) reversible photocontrol of hybridization by irradiation with appropriate wavelengths of light. We believe that photon-fueled DNA nanomachines driven by azobenzene-derivative molecular photon-fueled engines will be soon science rather than "science fiction".

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Various three-dimensional structures have been created on a nanometer scale using the self-assembly of DNA molecules. However, ordinary DNA structures breakdown readily because of their flexibility. In addition, it is difficult to control them by inputs from environments. Here, we construct robust and photocontrollable DNA capsules using azobenzenes. This provides a method to construct DNA structures that can survive higher temperatures and can be controlled with ultraviolet irradiation.

We synthesized various azobenzenes methylated at their ortho positions with respect to the azo bond for more effective photoregulation of DNA hybridization. Photoregulatory efficiency, evaluated from the change of T(m) (DeltaT(m)) induced by trans-cis isomerization, was significantly improved for all ortho-modified azobenzenes compared with non-modified azobenzene due to the more stabilized trans form and the more destabilized cis form. Among the synthesized azobenzenes, 4-carboxy-2',6'-dimethylazobenzene (2',6'-Me-Azo), in which two ortho positions of the distal benzene ring with respect to carboxyl group were methylated, exhibited the largest DeltaT(m), whereas the newly synthesized 2,6-Me-Azo (4-carboxy-2,6-dimethylazobenzene), which possesses two methyl groups on the two ortho positions of the other benzene ring, showed moderate improvement of DeltaT(m). Both NMR spectroscopic analysis and computer modeling revealed that the two methyl groups on 2',6'-Me-Azo were located near the imino protons of adjacent base pairs; these stabilized the DNA duplex by stacking interactions in the trans form and destabilized the DNA duplex by steric hindrance in the cis form. In addition, the thermal stability of cis-2',6'-Me-Azo was also greatly improved, but not that of cis-2,6-Me-Azo. Solvent effects on the half-life of the cis form demonstrated that cis-to-trans isomerization of all the modified azobenzenes proceeded through an inversion route. Improved thermal stability of 2',6'-Me-Azo but not 2,6-Me-Azo in the cis form was attributed to the retardation of the inversion process due to steric hindrance between lone pair electrons of the pi orbital of the nitrogen atom and the methyl group on the distal benzene ring.

A supra-photoswitch is designed for complete ON/OFF switching of DNA hybridization by light irradiation for the purpose of using DNA as a material for building nanostructures. Azobenzenes, attached to D-threoninols that function as scaffolds, are introduced into each DNA strand after every two natural nucleotides (in the form (NNX)n where N and X represent the natural nucleotide and the azobenzene moiety, respectively). Hybridization of these two modified strands forms a supra-photoswitch consisting of alternating natural base pairs and azobenzene moieties. In this newly designed sequence, each base pair is sandwiched between two azobenzene moieties and all the azobenzene moieties are separated by base pairs. When the duplex is irradiated by visible light, the azobenzene moieties take the trans form and this duplex is surprisingly stable compared to the corresponding native duplex composed of only natural oligonucleotides. On the other hand, when the azobenzene moieties are isomerized to the cis form by UV light irradiation, the duplex is completely dissociated. Based on this design, a DNA hairpin structure is synthesized that should be closed by visible light irradiation and opened by UV light irradiation at the level of a single molecule. Indeed, perfect ON/OFF photoregulation is attained. This is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nanodevices and other nanostructures.

A stable double helix involving alternating base pairs and azobenzene moieties was constructed. In this supramolecule, base pairs and azobenzenes are lined up one by one to form an interstrand-wedged motif: each base pair is sandwiched with two azobenzenes, and each azobenzene intercalates between two base pairs. This motif was formed by the hybridization of two modified DNA tethering multiple azobenzene moieties at a frequency of one azobenzene for every two nucleotides. Furthermore, this structure could be simply dismantled by UV light irradiation and reformed with the irradiation of visible light. By using this new duplex motif, construction of a variety of photoresponsive nanostructures and nanodevices is highly expected.

Two diastereomers were produced by the introduction of azobenzene-tethering prochiral linker (2,2-bis(hydroxymethyl)propionic acid) in the modified ODN, which had been used for the photoregulation of DNA functions. We found that this modified ODN with sequence 5'-...pNpXpN...-3' (p = phosphate; N = nucleoside; X = azobenzene residue) could be digested to pX (the phosphate at the 5' side of X was left) by an over excess of Phosphodiesterase I. By comparing the retention time of pX from the separated diastereomer with that of authentic R- or S-pX on chiral HPLC, absolute configuration could be easily determined.

A photoresponsive DNA nano-pincette was constructed by using azobenzene-modified DNA as materials. When the azobenzene-modified part hybridizes with its complementary sequence on the pincette, the duplex formation closes it. On the contrary, the pincette is opened after the formed duplex dissociates. Based on reversible photoswitching of this DNA hybridization, the pincette involving non-substituted azobenzene can be opened simply by UV light irradiation and closed by visible light irradiation. Interestingly, the operation can be reversed by using para-isopropyl group substituted azobenzene: visible light opens the pincette, and UV light closes it. In both cases, the azobenzene-modified part was attached to the pincette throughout the open/close operation, which makes single molecular operation possible. Furthermore, the operation can be repeated many times without any decrease of the cycling efficiency and no DNA waste was produced.

The introduction of methyl groups into two ortho positions (2' and 6' positions) of the same benzene ring in an azobenzene remarkably raised both its photoregulation ability and the thermal stability of the cis-form.

We constructed a photoresponsive T7 promoter by tethering two 2,6-dimethyl azobenzenes (4-(2,6-dimethyl-phenlylazo)-benzoic acids) via D-threoninol linkers. Under UV light irradiation, the rate of transcription with T7 RNA polymerase (RNAP) on the photoresponsive promoter was 10-fold faster than that under visible light irradiation. Kinetic analysis revealed that K(m) of cis-cis-promoter (both of the introduced azobenzenes are in cis-form) with T7 RNAP was more than 60 times larger than that of trans-trans-form, indicating that the high photoregulatory efficiency was obtained mainly due to the remarkable difference in their affinity with RNAP. By attaching a photoresponsive promoter to the GFP gene, we also showed that photoregulation of gene expression became practicable.

A phosphoramidite monomer bearing an azobenzene is synthesized from D-threoninol. Using this monomer, azobenzene moieties can be introduced into oligodeoxyribonucleotide (DNA) at any position on a conventional DNA synthesizer. With this azobenzene-tethered DNA, formation and dissociation of a DNA duplex can be reversibly photo-regulated by cis-trans isomerization of the azobenzene. When the azobenzene takes a trans-form, a stable duplex is formed. After isomerization of the trans-azobenzene to its cis-form by UV-light irradiation (300 nm < lambda < 400 nm), the duplex can be dissociated into two strands. The duplex is reformed on photo-induced cis-trans isomerization (lambda > 400 nm). The introduction of azobenzenes into the T7 promoter at specific positions also efficiently and reversibly photo-regulates transcription by T7-RNA polymerase. The reversible regulation can be repeated many times without causing damage to the DNA or the azobenzene moiety. These procedures take approximately 10 d to complete.

Previously, photoregulation of DNA hybridization was achieved by introducing nonsubstituted azobenzene via a D-threoninol linker: DNA duplex formed (ON) after visible light irradiation (planar trans-form), whereas the duplex dissociated (OFF) after UV light irradiation (non-planar cis-form). In this study, for more efficient photoregulation of DNA functions, the reverse switch that can turn on duplex formation with UV, and turn off it with visible light irradiation was designed. When para-isopropylazobenzene (p-(i)PrAzo) was introduced into DNA via a L-thereoninol linker, the photoswitching direction was completely reversed: the duplex involving non-planar cis-p-(i)PrAzo was much more stable than that involving planar trans-form.

New alternating hetero aggregates ((")comb-type(") hetero aggregates) were successfully prepared by hybridizing two single-stranded DNAs, each of which tethered different homo-dye aggregates in the centre of the strand. When excitonically inert dyes (Methyl Red and azobenzene) were alternately assembled, broadening of the spectrum was observed. On the other hand, alternating aggregates of Methyl Red and Naphthyl Red showed substantial narrowing of the band. Although the peak maxima of these two dyes were different, strong exciton coupling occurred.

Azobenzene was additionally introduced into side chain of T7 promoter for the photocontrol of transcription reaction by T7 RNA polymerase (T7 RNAP). When a single azobenzene molecule was introduced into the T7 promoter either at the loop-binding region of the RNAP (-7 to -11 position) or at the unwinding region (-1 to -4 position), transcription was suppressed in the trans-form but proceeded faster in the cis-form. The amount of transcripts after UV irradiation with respect to that in the dark was 1.5-2.0-fold. Kinetic analysis of the transcription reaction revealed that the photoregulatory mechanism was different in these positions. The photoisomerization of an azobenzene at the loop-binding region primarily affected Km. On the other hand, the isomerization of an azobenzene at the unwinding region mainly affected kcat. Still more clear-cut photoregulation was achieved when two azobenzenes were introduced into both loop-binding and unwinding regions, respectively: transcription proceeded 7.6-fold faster after UV irradiation than that in the dark. This synergistic effect was observed only when two azobenzenes were introduced into these two different regions, respectively, and introduction of them into the same loop-binding region drastically lowered the transcription activity. The cooperation of two azobenzenes at loop-binding and unwinding regions would contribute to the clear-cut photoregulation of transcription.

An ordered dye cluster of Methyl Reds was formed in double-stranded DNA by hybridizing two complementary DNA-dye conjugates, each involving a Methyl Red moiety on a threoninol linker and a 1,3-propanediol spacer arranged alternately in the middle of the DNA sequence. In the duplex, Methyl Reds from each strand were axially stacked antiparallel to each other, as determined from NMR analysis. This clustering of Methyl Reds induced distinct changes in both UV/Vis and CD spectra. Single-stranded DNA-Methyl Red conjugates on D-threoninol linkers and (1,3-propanediol) spacers exhibited broad absorption spectra with lambda(max) at around 480 nm, and almost no CD was observed at around the absorption maximum of Methyl Red. However, as Methyl Reds were clustered by hybridization, lambda(max) shifted towards shorter wavelengths with respect to its monomeric transition. This hypsochromic shift increased as the number of Methyl Red molecules increased. Furthermore, a positive couplet was also strongly induced here. These dye clusters are H-aggregates, in which molecular excitons are coupled. The positive couplet demonstrates that the clusters on D-threoninol form a right-handed helix. In contrast, the induced CD became much weaker with Methyl Red on L-threoninol, which intrinsically prefers counterclockwise winding. Thus, mutual orientation of the stacked dye molecules was controlled by the chirality of the linker.

We have synthesized azobenzene-tethered DNAs and have successfully photo-regulated various DNA functions. In the present study, we synthesized azobenzenes substituted with methyl group for still more effective photo-regulation of DNA hybridization. In trans-form, mono substituted azobenzene at ortho position stabilized the DNA duplex more efficiently than the other mono-substituted ones. In contrast, melting temperature (T(m)) for 2-methylazobenzene was lower in cis-form. As a result, change of T(m) (DeltaT(m)) induced by trans-cis isomerization became larger than that of unmodified azobenzene. Furthermore, di-substituted azobenzene at both ortho positions exhibited even larger DeltaT(m). Quite interestingly, thermal cis-to-trans isomerization of this azobenzene was about 10-fold slower than that of unmodified one. Thus, introduction of methyl group at 2, 6 positions raised both photo-regulatory activity and thermal stability of cis-form.

Multiple azobenzenes were introduced into the oligonucleotide on D-threoninol for the clear-cut photo-regulation of the formation and dissociation of DNA duplex. When azobenzenes took trans form, introduction of multiple azobenzenes (azobenzenes:nucleobases = 1:2) did not interfere with duplex formation at all compared with their native duplex. In contrast, melting temperature (Tm) uniformly decreased with the number of azobenzenes when they took cis-form. As a result, clear-cut photo-regulation of the duplex formation was attained under physiological conditions. Furthermore, nearest-neighbor parameters for these azobenzene-tethered oligonucleotides were obtained. It was found that Tms estimated from these parameters well coincided with those of measured ones.

RNA digestion by RNase H, which is responsible for the antisense effect, was efficiently photoregulated by use of the duplex of azobenzene-tethered sense DNA and native antisense DNA. In the dark, RNA digestion was suppressed because antisense DNA was strongly hybridized with azobenzene-tethered sense DNA, and accordingly RNA was isolated. On UV irradiation, antisense DNA was released from the azobenzene-tethered DNA due to the trans-to-cis isomerization and hybridized with RNA, which was digested by RNase H.

We have already reported that transcription reaction by phage T7 or SP6 RNA polymerase (RNAP) can be reversibly photo-regulated with azobenzene-tethered promoter. Transcription reaction proceeded faster by UV irradiation than visible light irradiation. In the present study, binding of T7 RNAP to its azobenzene-tethered promoter was directly monitored by use of affinity biosensor. When azobenzene-tethered T7 promoter was immobilized on the sensor surface, response based on the binding of RNAP increased by UV light irradiation rather than by visible light irradiation. Thus, photo-regulation of transcription reaction by azobenzene-tethered promoter was attributed to the change of binding property of RNAP to the promoter by trans-cis isomerization of azobenzene.

Based on a previous study of photo-regulation of transcription by T7 RNA polymerase, the regulatory effect of azobenzene on in vitro green fluorescent protein (GFP) production was studied by introduction of a D-threoninol-tethered p-azobenzene into the non template strand of the T7 promoter in a GFP template. By measurement of the resulting GFP fluorescence intensity after the in vitro transcription/translation coupled reaction, it is found that photo-regulation of GFP gene expression can be achieved in a position-dependent manner by trans-cis isomerization of azobenzene upon UV irradiation.

Two diastereomers of a photoresponsive oligodeoxyribonucleotide tethering a trans-azobenzene, based on the chirality of the central carbon of a diol linker, were separated by reversed-phase HPLC. On the basis of 2D NMR analysis, absolute configurations of the diastereomers alpha and beta (tentatively designated from differences in their retention time) were determined as R- and S-forms, respectively. For both diastereomers, their NMR-determined duplex structure showed that trans-azobenzene intercalates between base pairs, because distinct NOEs were observed between the protons of azobenzene and those of the adjacent base pairs, such as with the imino protons and methyl protons of thymine. The melting temperatures of both duplexes were higher than that of the corresponding native duplex, which contained no azobenzene residue, due to the intercalated trans-azobenzene stabilizing the duplex by a stacking interaction. Between these two diastereomers, differences in T(m) were also found: the melting temperature of the R-form duplex (alpha-isomer) was higher than that of the S-form (beta-isomer). On the basis of the NMR-determined structure, this difference was attributed to the fact that the S-form (beta isomer) causes more stress forming the duplex than does the R-form (alpha isomer) due to disturbances of the right-hand helix.

Methyl Red H aggregate of predetermined size is successfully synthesized from the DNA conjugate involving multiple Methyl Red moieties in sequence. In the single stranded state, hypsochromicity monotonically increases with the number of incorporated dyes: the peak maximum of the conjugate involving six Methyl Reds appears at 415 nm, and the shift is as great as 69 nm (3435 cm(-)(1)) with respect to the monomeric transition. This large hypsochromicity accompanied by the narrowing of the band clearly demonstrates that H aggregate is formed in the single strand. H aggregation is further promoted at higher ionic strength. Upon addition of complementary DNA below the T(m), however, this H band disappears and a new peak appears at 448 nm, indicating that aggregated structure is changed by the duplex formation. This spectral change is completely reversible so that the H band at 415 nm appears again above T(m). Thus, aggregated structure can be reversibly controlled by the formation and dissociation of the DNA duplex.

Multiple azobenzene moieties were tethered to DNA on D-threoninol linker, and formation and dissociation of DNA duplex could be reversibly photo-regulated either by irradiating UV or visible light. With this photo-responsive DNA, RNase H reaction was also successfully photo-regulated.

Effective photo-regulation of transcription reaction by SP6 RNA polymerase (RNAP) was achieved with photo-responsive SP6 promoter tethering two azobenzenes. With one azobenzene in either TATA or RNAP binding region of the SP6 promoter, photo-regulation activity was very small. But when two azobenzenes were introduced into both regions, efficient photo-regulation of transcription was attained: transcription proceeded 3.5 fold faster under UV irradiation than under dark.

Formation and dissociation of DNA triplex are reversibly photoregulated by cis <--> trans isomerization of the azobenzene tethered to the third strand. When the azobenzene takes the trans from, a stable triplex is formed. Upon the isomerization of trans-azobenzene to its cis form by UV light irradiation (300 < lambda < 400 nm), however, the modified oligonucleotide is removed from the target duplex. The triplex is re-formed on photoinduced cis --> trans isomerization (lambda > 400 nm). The photoregulating activity significantly depends on the position of azobenzene in the third strand, as well as on the geometric position (meta or para) of its amido substituent. For m-amidoazobenzene, the photoregulation is the most effective when it is tethered to the 5'-end of the third strand. However, p-amidoazobenzene should be introduced into the middle of the strand for effective regulation. In the optimal cases, the change of T(m) of the triplex, caused by the cis <--> trans isomerization of azobenzene, is greater than 30 degrees C. UV-visible and CD spectroscopy, as well as computer modeling studies, clearly demonstrate that the trans-azobenzene intercalates between the base pairs in the target duplex and thus stabilizes the triplex by stacking interactions. On the other hand, nonplanar cis-azobenzene destabilizes the triplex due to its steric hindrance against the adjacent base pairs.

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials, and nanofactories. Present here is an entirely light‐induced DNA walker based on orthogonal photocontrol. Implementing two azobenzene derivatives, S‐DM‐Azo and DM‐Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non‐autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back‐and‐forth directions as desired, and is regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non‐autonomous progressive molecular motors, ultimately facilitating their application at the nanoscale. [ABSTRACT FROM AUTHOR]

Interstrand H-aggregates of cationic dyes have been prepared by hybridization of two DNA-dye conjugates involving a Naphthyl Red moiety and spacer alternately in the middle of the sequence. At pH 5.0 where Naphthyl Red is positively charged, a single-stranded DNA-Naphthyl Red conjugate involving three dye groups and three spacer residues exhibited a broad absorption spectrum with λmax at around 530nm. However, hybridization of two DNA-Naphthyl Red conjugates that were complementary provided different spectra: a much narrower absorption band appeared at 507nm, that is 23nm shorter than the single-stranded conjugates. This spectroscopic behavior indicates that the dyes in the duplex are H-aggregated and excitons are strongly coupled in the aggregate. In addition to the appearance of the narrow H-band, the melting temperature dramatically increased by the H-aggregation of the stacked cationic dyes compared with that of the natural duplex without dye and spacer residues. Thus, the positive charge on the stacked dyes did not interfere with the duplex formation by electrostatic repulsion, but in fact promoted the hybridization. [ABSTRACT FROM AUTHOR]