Hidenori Nishioka

Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription

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 < λ < 400 nm), the duplex can be dissociated into two strands. The duplex is reformed on photo-induced cis–trans isomerization (λ > 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. NOTE: In Figure 4 of the version of this article originally published online, the base sequence of the oligonucleotide was incorrect. The figure has been replaced in all versions of the article.

A DNA Nanomachine Powered by Light Irradiation

Over the past decade, DNA has been widely used for the ACHTUNGTRENNUNGdevelopment of nanomaterials because it undergoes highly ACHTUNGTRENNUNGsequence-specific hybridization and forms a highly regular double-helical structure with suitable flexibility. DNA is probably one of the most promising biomolecules for future applications in nanotechnology and materials science. Many 2D and 3D nanostructures with determined shapes and geometries have been reported recently in which DNA is used as the building blocks and mortar. 6, 7] More excitingly, several types of DNA nanomachines, fuelled with DNA oligonucleotides or other molecules such as intercalators and metal ions, have been constructed. During these 10 years of development, substantial progress has been made in the design of DNA-based devices such as tweezers, walkers, and gears, which can perform mechanical functions such as scission, directional motion, or rolling. The prospects of this field are extraordinarily promising, and several valuable applications of DNA nanomachines as sensors, transporters, and drug-delivery systems have also been reported. For most of the DNA nanomachines constructed so far, oligonucleotides have been generally used as the fuel. In many of these systems, the mechanical motion was usually carried out by hybridization of one DNA fuel molecule to target sequences followed by its removal with another DNA sequence that is completely or partially complementary to the first. Yurke et al. demonstrated the first DNA machine that functioned as “tweezers” fuelled by two strands of DNA with tailored complementarity. As the energy for operating these DNA nanomachines is produced by a strand-exchange strategy, a DNA duplex is produced as a waste product in every working cycle. Thus, the operating efficiency decreases gradually with the accumulation of “wastes”. A new strategy is therefore required to overcome this problem for the further development of DNA nanotechnology. Over the past decade, we have developed a series of photoresponsive DNAs by covalently tethering azobenzene moieties onto the DNA strand. Hybridization of these photoresponsive DNAs to single-stranded DNA (to form duplexes), RNA (to form DNA–RNA hybrids), or double-stranded DNA (to form triplexes) can be efficiently switched “on” and “off” by simply irradiating with UV and visible light. This is based on the following mechanism: the planar trans-azobenzene intercalates between adjacent base pairs and stabilizes the duplex or triplex structure by stacking interactions, whereas the nonplanar cisazobenzene destabilizes it by steric hindrance. The successful photoregulation of primer elongation, transcription, and RNase H activity have also been demonstrated with photoresponsive DNAs. Photoregulation efficiency can be amplified by the introduction of multiple azobenzene residues onto the DNA. For example, nine azobenzene groups were introduced onto a DNA strand 20 nucleotides (nt) in length, and the clearcut photoswitching of DNA duplex formation was observed without loss of sequence specificity. Photoregulation of the opening of a DNA hairpin as a simple nanomachine by invasion of a DNA opener was also recently demonstrated. All these results prompted us to propose a new strategy for building photon-fuelled DNA nanomachines that are “environmentally friendly” without producing DNA waste during operation. Herein we report a simple, inexpensive, clean, and long-lived photoresponsive DNA nanomachine that can be operated continuously by reversibly photoswitching its mechanical motion with light irradiation. A photoresponsive DNA machine composed of four strands (A, B, C, and F) was designed based on the DNA-fuelled tweezers reported by Yurke et al. , as illustrated in Figure 1. Strand A is hybridized with strands B and C to form two stiff doublestranded arms that are 22 base pairs long and sufficiently stable at the operating temperature. Tetrachlorofluorescein (TET) and carboxytetramethylrhodamine (TAMRA) were attached respectively to the 5’ and 3’ ends of strand A. When strand F is hybridized with the overhangs of strands B and C (left side of Figure 1A), the tweezers are closed, and the fluorescence emission from TET at ~540 nm (lex=514.5 nm) is quenched by resonant intramolecular energy transfer to TAMRA due to the close proximity of these two dyes. However, when strand F is dissociated (right side of Figure 1A), the tweezers are open, and the fluorescence from TET is recovered. Here, 12 azobenzene moieties are introduced onto a 32-ntlong strand F (F12X) so that opening and closure of the tweezers can be photo-controlled by the dissociation and hybridization of strand F (F12X) with B and C. What we expect is as follows: the tweezers are closed after visible light irradiation (azobenzene cis!trans isomerization), whereas UV light irradiation (trans!cis isomerization) opens them due to the destabilization effect of cis-azobenzene. When strands A, B, and C were mixed in the absence of strand F at 50 8C, strong fluorescence from TET was observed because the tweezers were completely open (Figure 2: b). In the presence of Fn (the native form of strand F), however, the fluorescence decreased dramatically because the tweezers [a] Prof. Dr. H. Asanuma Graduate School of Engineering, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan) Fax: (+81)52-789-2528 E-mail : asanuma@mol.nagoya-u.ac.jp [b] Prof. Dr. X. Liang, H. Nishioka, N. Takenaka Core Research for Evolution Science and Technology (CREST) Japan Science and Technology Agency (JST) Kawaguchi, Saitama 332-0012 (Japan) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

An interstrand-wedged duplex composed of alternating DNA base pairs and covalently attached intercalators

An interstrand-wedged duplex involving alternating base pairs and covalently attached intercalators viaD-threoninols was constructed. In this novel duplex structure, natural DNA base pairs and artificially introduced planar molecules such as azobenzene derivatives are lined up one by one. Although each base pair is sandwiched by two intercalators and vice versa, the duplex is extremely stable compared with the corresponding native DNA duplex. Analysis by 1H-NMR showed that all of the expected internal base pairs are strongly formed even when the base pairs are sandwiched between azobenzene moieties. More interestingly, the stability of this new duplex structure decreased greatly when a mismatched base pair was introduced, indicating that this interstrand-wedged duplex has a high sequence specificity. Furthermore, the structure became very unstable when azobenzene moieties were photoisomerized to the cis form. It is expected that this new duplex motif will be very useful for the construction of a variety of functional nanostructures and nanodevices. The concept proposed here is a novel combination of the specificity of DNA hybridization and the versatility of synthesized organic molecules.

Molecular Design for Reversing the Photoswitching Mode of Turning ON and OFF DNA Hybridization

A new photoswitch for DNA hybridization involving para-substituted azobenzenes (such as isopropyl- or tert-butyl-substituted derivatives) with L-threoninol as a linker was synthesized. Irradiation of the modified DNA with visible light led to dissociation of the duplex owing to the destabilization effect of the bulky substituent on the trans-azobenzene. In contrast, trans-to-cis isomerization (UV light irradiation) facilitated duplex formation. The direction of this photoswitching mode was entirely reversed relative to the previous system with an unmodified azobenzene on D-threoninol whose trans form turned on the hybridization, and cis form turned it off. Such reversed and reversible photoswitching of DNA hybridization was directly demonstrated by using fluorophore- and quencher-attached oligonucleotides. Furthermore, it was revealed that the cis-to-trans thermal isomerization was greatly suppressed in the presence of the complementary strand owing to the formation of the more-stable duplex in the cis form.

Light driven open/close operation of an azobenzene-modified DNA nano-pincette

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.

Preparation of Photoresponsive DNA Tethering Ortho‐Methylated Azobenzene as a Supra‐Photoswitch

This unit describes synthetic procedures of photoresponsive DNA via a phosphoramidite monomer composed of D‐threoninol as a scaffold and 4‐carboxy‐2′,6′‐dimethylazobenzene or 4‐carboxy‐2′‐methylazobenzene that works as a photoswitch more efficiently than previous nonmodified azobenzene (4‐phenylazobenzoic acid). With these newly modified‐azobenzenes, photoregulatory efficiency of DNA hybridization can be greatly improved. Furthermore, thermal stability of cis‐azobenzene of 4‐carboxy‐2′,6′‐dimethylazobenzene remarkably increases compared with the previous non‐modified azobenzene. Curr. Protoc. Nucleic Acid Chem. 46:4.45.1‐4.45.18.

Line up base pairs and intercalators one by one in a stable duplex.

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.