Hiroyuki Asanuma

Photoregulation of the Formation and Dissociation of a DNA Duplex by Using the cis-trans Isomerization of Azobenzene.

The duplex-forming activity of an oligonucleotide has been photoregulated by making use of the isomerization of an azobenzene moiety in the side chain. When the azobenzene moiety is isomerized from the trans form to the cis form upon photoirradiation, the melting temperature of the duplex between the oligonucleotide and its complementary counterpart is significantly lowered, and the duplex is largely dissociated into two single-stranded oligonucleotides (shown schematically).

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.

Molecular imprinting of cyclodextrin in water for the recognition of nanometer-scaled guests

Acryloyl-cyclodextrins were synthesized as functional vinyl monomers, and various antibiotics and oligopeptides were molecularly-imprinted to them in water. The imprinting promoted the binding activity toward the template compared with non-imprinted polymer. The imprinting effect was eminent when the template involved more than two hydrophobic residues on a rigid molecular-frame. From Langmuir analysis, the promotion of binding by the imprinting was attributed to the increase in the binding constant, not increase in the number of binding sites. The present imprinting also provided eminent guest selectivity including enantioselectivity.

Tailor‐Made Receptors by Molecular Imprinting

Molecular recognition is becoming increasingly important in both research and industry (e. g. water purification). This review focuses on molecular imprinting with cyclodextrins—highly useful because of their hydrophilic exterior and hydrophobic cavity—including the very effective strategy adopted by the authors (see Figure): Several host species are assembled to form a tailor-made guest complex with extremely exclusive selectivity.

A supra-photoswitch involving sandwiched DNA base pairs and azobenzenes for light-driven nanostructures and nanodevices.

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.

Photocontrol of DNA Duplex Formation by Using Azobenzene-Bearing Oligonucleotides

The duplex‐forming activities of oligonucleotides can be photomodulated by incorporation of an azobenzene unit. Upon isomerizing the trans‐azobenzene to the cis form by irradiation with UV light, the Tm value of the duplex (with the complementary DNA) is lowered so that the duplex is dissociated. The duplex is formed again when the cis‐azobenzene is converted to the trans‐azobenzene by irradiation with visible light. The photoregulation is successful irrespective of the position of the azobenzene unit in the oligonucleotides. The trans‐azobenzene in the oligonucleotides intercalates between two DNA base pairs in the duplexes and stabilizes them because of a favorable enthalpy change. The nonplanar structure of a cis‐azobenzene is unfavorable for such an interaction. These photoresponsive oligonucleotides are promising candidates for the regulation of various bioreactions.

Robust and photocontrollable DNA capsules using azobenzenes.

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.

Photoregulation der Bildung und Dissoziation eines DNA‐Duplexes durch cis‐trans‐Isomerisierung einer Azobenzoleinheit

Die Neigung eines Oligonucleotids zur Duplexbildung wurde photoreguliert, indem die Isomerisierung einer Azobenzoleinheit in der Seitenkette genutzt wurde. Nach der Isomerisierung der Azobenzoleinheit von der trans- zur cis-Form durch Bestrahlung liegt die Schmelztemperatur des Duplexes aus dem Oligonucleotid und seinem komplementaren Gegenstuck wesentlich niedriger, und der Duplex ist weitgehend in zwei Einzelstrang-Oligonucleotide dissoziiert (schematisch gezeigt).

Photo-responsive oligonucleotides carrying azobenzene in the side-chains

Abstract The title oligonucleotides were prepared by using a new phosphoramidite monomer. The cis-trans isomers with respect to the stereochemistry of the azobenzene residue, obtained on photo-irradiation, were completely resolved by reversed-phase HPLC. The physicochemical properties of these oligonucleotides were significantly changed by the photo-induced isomerization.