Faculty Profiles - ASANUMA, Hiroyuki

Papers - ASANUMA, Hiroyuki

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An efficient FRET between pyrene and perylene assembled in a DNA duplex and its potential for discriminating single base changes Reviewed

Kashida, H.; Takatsu,T.; Sekiguchi, K.; H. Asanuma

Chem. Eur. J. Vol. 16 page： 2479-2486 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

In order to increase the apparent Stokes' shift of perylene, pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to achieve the efficient fluorescence resonance energy transfer (FRET) from pyrene to perylene. Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between the dyes. Accordingly, donors and acceptors could be accumulated inside the DNA without forming undesired excimer/exciplex. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 460 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high apparent FRET efficiency.

Construction of photoresponsive RNA for photoswitching RNA hybridization Reviewed

Ito, H.; Liang, X.G.; Nishioka, H.; Asanuma, H.

Org. Biomol. Chem. page： in press 2010

Robust and photo-controllable DNA capsules using azobenzenes Reviewed

Tanaka, F.; Mochizuki, T.; Liang, X.G.; Asanuma, H.; Tanaka, S.; Suzuki, K.; Kitamura, S.; Nishikawa, A.; Ui-Tei, K.; Hagiya, M

Nano Lett. Vol. 10 page： 3560-3565 2010

Unexpectedly stable artificial duplex from flexible acyclic threoninol. Reviewed

Asanuma, H.; Toda, T.; Murayama, K.; Liang, X.G.; Kashida, H.

J. Am. Chem. Soc. Vol. 132 page： 14702-14703 2010

Insulator base pairs for lighting-up perylenediimide in a DNA duplex Reviewed

Hiromu Kashida,Koji Sekiguchi,and Hiroyuki Asanuma

Chem. Eur. J. Vol. 16 page： 11554-11557 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Perylenediimide (PDI) is highly quenched by nucleobases, which greatly restricts its application as a fluorescent probe. Here, we propose “insulator base pairs" tethering cyclohexane ring through D-threoninol. When “insulator base pairs" were inserted between PDI and nucleobases, the quantum yield of PDI drastically increased several thousand-fold. The “insulator base pairs" reported here also have the potential to increase the quantum yields of other fluorophores.

Coherent Quenching of a Fluorophore for the Design of a Highly Sensitive In-Stem Molecular Beacon. Reviewed

Yuichi Hara, Taiga Fujii, Hiromu Kashida, Koji Sekiguchi, Xingguo Liang, Kosuke Niwa, Tomokazu Takase, Yasuko Yoshida, and Hiroyuki Asanuma

Angew. Chem. Int. Ed. Vol. 49 page： 5502-5506 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Excitonic interaction (coherency) was utilized to design a highly sensitive In-Stem molecular beacon (ISMB) in which both a fluorophore and a quencher on D-threoninols are incorporated into the stem region as a pseudo “base-pair". A systematic study indicated that minimization of the difference of max between the fluorophore and the quencher maximized quenching efficiency due to maximization of coherency. A highly sensitive ISMB could be prepared using the excitonically optimized pair of Cy3 and modified Methyl Red.

Accumulation of fluorophores into DNA duplexes to mimic the properties of quantum dots Reviewed

Hiromu Kashida, Koji Sekiguchi, Xingguo Liang, Hiroyuki Asanuma

J. Am. Chem. Soc. Vol. 132 page： 6223-6230 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

By using perylene and pyrene as fluorophores, we have designed various fluorophore assemblies that mimic inorganic quantum dots in showing a high emission intensity, a large Stokes' shift, and a modulated emission maximum. For this purpose, we utilized two kinds of duplex motifs with D-threoninols as scaffolds: cluster and interstrand-wedged motifs. In the cluster motif, fluorophores are introduced into both strands to produce tentative pseudo-“base-pairs", in which the dyes strongly interact with each other and form dimers, trimers or hexamers. In the interstrand-wedged motif, a base-pair is inserted between the fluorophores to suppress their direct interaction. These two motifs were applied to accumulate dyes within a DNA duplex depending on their emission properties. Since pyrene exhibits strong excimer emission, the emission at 500 nm of a pyrene cluster motif strongly increased as the number of accumulated dyes increased, whereas interstrand-wedged motif quenched pyrene monomer emission. In contrast, assembled perylenes, which are mostly quenched by dimerization, showed intense monomer emission in the interstrand-wedged motif whereas perylene cluster motifs strongly suppressed perylene emission. These two motifs were then applied to the hetero-assembly of pyrenes and perylenes. Both a large Stokes' shift and a modulation of the emission maximum, which are also characteristics of inorganic quantum dots, were successfully realized using fluorescent resonance energy transfer (FRET) and exciplex formation. These fluorophore assemblies thus obtained could be enzymatically ligated to longer DNA, demonstrating that this technique has the potential to be a versatile labeling agent for biomolecules.

*Photoregulation of DNA transcription by using photoresponsive T7 promoter and clarification of its mechanism Reviewed

Xingguo Lianga, Ryuji Wakuta, Kenta Fujioka, Hiroyuki Asanuma

FEBS Journal Vol. 277 page： 1551-1561 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Using photoresponsive T7 promoters tethering two 2-methylazobenzenes or 2,6-dimethylazobenzenes, highly efficient photoregulation of DNA transcription was attained. Under UV-A 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 non-modified azobenzene (Azo) and 2,6-dimethylazobenzene (DM-azo) at the two positions, respectively, and by utilizing the different cis-to-trans thermal stability between cis Azo and cis DM-azo, four species of T7 promoter (cis-cis, trans-cis, cis-trans, and trans-trans) were obtained. The four species showed transcriptional activity in the order of cis-cis > cis-trans > trans-cis > trans-trans. Kinetic analysis revealed that the Km for the cis-cis promoter (both of the introduced azobenzene derivatives were in the cis form) and T7 RNA polymerase was 68 times larger than that of the trans-trans form, indicating that high photoregulatory efficiency was mainly due to a remarkable difference in affinity with RNA polymerase. The present approach is promising for the creation of biological tools for artificially controlling gene expression, and as a photo-controlled system for supplying RNA fuel for RNA-powered molecular nanomachines.

*A light-driven DNA nanomachine for efficiently photoswitching RNA digestion Reviewed

Zhou M.G.; Liang, X.G.; Mochizuki, T.; Asanuma, H.

Angew. Chem. Int. Ed. Vol. 49 page： 2167-2170 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

A machine-like photoresponsive DNA enzyme was constructed that can work for us at a single molecule level. The complete ON-OFF photoswitching of RNA digestion was realized by photoregulating the topological structure of DNAzyme/RNA complex. The interstrand-wedged duplex was used as the supra-photoswitch.

Effect of the ortho modification of azobenzene on the photoregulatory efficiency of DNA hybridization and thermal stability of its cis-form Reviewed

Nishioka H.; Liang, X.G.; Asanuma, H.

Chem. Eur. J. Vol. 16 page： 2054-2062 2010

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

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 Tm (Tm) induced by trans-cis isom-erization, was significantly improved for all ortho-modified azobenzenes compared with non-modified azoben-zene due to the more stabilized trans-form and 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 methy-lated, exhibited the largest Tm, whereas 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 im-provement of Tm.

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

Liang, X.G.; Mochizuki T.; Nishioka H.; Asanuma, H.

Nucleic Acids Symp. Ser. Vol. 53 page： 189-190 2009.11

Authorship：Lead author Language：English

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.

Efficient energy transfer from pyrene to perylene assembled inside DNA duplex

Kashida, H.; Takatsu, T.; Asanuma, H.

Nucleic Acids Symp. Ser., Vol. 53 page： 29-30 2009.11

Authorship：Lead author Language：English

Pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to increase the apparent Stokes' shift of perylene. Plural Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between donors and acceptors in order to suppress undesired interactions between them. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 495 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high FRET efficiency (>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R0) of the donor pyrene.

*Analysis of Coherent Heteroclustering of Different Dyes by Use of Threoninol-Nucleotides for Comparison with the Molecular Exciton Theory Reviewed

Taiga Fujii, Hiromu Kashida, Hiroyuki Asanuma

Chem. Eur. J. Vol. 15 page： 10092-10102 2009.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

To test the molecular exciton theory for heterodimeric chromophores, various heterodimers and clusters, in which two different dyes were stacked alternately, were prepared by hybridizing two oligodeoxyribo-nucleotides (ODNs), each of which tethered a different dye on D-threoninol at the center of the strand. NMR analyses revealed that two different dyes from each strand were stacked antiparallel to each other in the duplex, and were located adjacent to the 5'-side of a natural nucleobase. Spectroscopic behaviors of these heterodimers were systematically examined as a function of the difference in the wavelength of the dye absorption maxima (max). We found that the absorption spectrum of the heterodimer was significantly different from that of the simple sum of each monomeric dye in the single-strand. When azobenzene and Methyl Red, which have max at 336 nm and 480 nm in the single-strand, respectively, (max = 144 nm), were assembled on ODNs, the band derived from azobenzene exhibited a small hyperchromism whereas the band from Methyl Red showed hypochromism and both bands shifted to a longer wavelength (bathochromism). These hyper- and hypochromisms were further enhanced in a heterodimer derived from 4'-methylthioazobenzene and Methyl Red that had a much smaller max (82 nm) (max = 398 and 480 nm in the single-strand, respectively). With a combination of 4'-dimethylamino-2-nitroazobenzene and Methyl Red, which had an even smaller max (33 nm), a single sharp absorption band that was apparently different from the sum of the single-stranded spectra was observed. These changes in the intensity of the absorption band could be explained by the molecular exciton theory that has been mainly applied to the spectral behavior of H- and/or J-aggregates composed of homo dyes. However, the bathochromic band shifts observed at shorter wavelengths did not agree with the hypsochromism predicted by the

*In-Stem Molecular Beacon containing a Pseudo Base Pair of Threoninol Nucleotides for Removal of Background Emission Reviewed

Hiromu Kashida, Tomohiko Takatsu, Taiga Fujii, Koji Sekiguchi, Xingguo Liang, Kosuke Niwa, Tomokazu Takase, Yasuko Yoshida, and Hiroyuki Asanuma

Angew. Chem. Int. Ed. Vol. 48 page： 7044-7047 2009.9

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

An in-stem molecular beacon (IS-MB), which tethers perylene and anthraquinone in the stem region via D-threoninol, effectively detected target sequences. This design of IS-MB is also applicable to the discrimination of a one-base deletion mutant from wild type (full-match) without background emission.

Photoisomerization dynamics study on cis-azobenzene derivative using ultraviolet-to-visible tunable femtosecond pulses Reviewed

Yamaguchi, N.; Nagasawa, N.; Igarashi, K.; Sekikawa, T.; Asanuma, H.; Yamashita, M.

Appl. Surf. Sci. Vol. 255 page： 9864-9868 2009.7

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

We performed the transient absorption measurement and the first rate equation (RE) analysis for cis isomer of 4-carboxy-20,60-dimethylazobenzene to clarify the quantitative difference between the photoisomerization process and the thermal relaxation process from the SC
1 excited state. The RE analysis enabled us to determine the cis-to-trans photoisomerization rate per each pump pulse to be 3% under the condition of the 430 nm, 150 fs pump pulse with energy of 200 nJ. Moreover, the signal due to the yielded trans molecules appearing in the transient absorption was assigned from the following observed result: the transient absorbance change at the 380 nm probe mostly decreased within 300 fs after the 430 nm pulse pumping and then slowly decreased to zero, while the absorbance change at the 350 nm probe had a positive constant component in the over one picosecond time region. The RE analysis showed
that this constant component is due to the yielded transmolecules, and its positive value is due to the fact
that the absorption cross-section of the ST 0 -to- ST
2 transition in their trans molecules is larger than that of
the SC0 -to- SC 2 transition in the original cis molecules.

Positively charged base surrogate for highly stable “base-pairing" through electrostatic and stacking interactions. Reviewed

Hiromu Kashida, Hidehiro Ito, Taiga Fujii, Takamitsu Hayashi, Hiroyuki Asanuma

J. Am. Chem. Soc. Vol. 131 page： 9928-9930 2009.7

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

“Base pairs" of cationic dyes (p-methylstilbazole) were incorporated into oligodeoxyribonucleotides (ODNs). This “base pair" greatly stabilized the duplex through electrostatic and stacking interactions. The melting temperature of modified ODN was higher than those of neutral dyes and native base pairs. Further stabilization of the duplex was observed when the number of cationic dyes increased.

*A Supra-Photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and Nanodevices Reviewed

Liang, X.G.; Mochizuki, T.; Asanuma, H.

Small Vol. 5 ( 15 ) page： 1761-1768 2009.6

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

A supra-photoswitch was 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, were 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 formed 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 was 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 was attained. This design is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nano devices and other nanostructures.

Construction of Photon-Fueled DNA Nanomachines by Tethering Azobenzenes as Engines Reviewed

Liang, X.G.; Nishioka, H.; Takenaka, N.; Asanuma, H.

Lecture Note in Computer Science Vol. 5347 page： 21-32 2009

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Nanoscale DNA tweezers operated by photo-irradiation were constructed by using azobenzene-modified DNA as materials. The azobenzenes that can photoisomerize between trans and cis form were used as the engines to open and close the tweezers. The work principle is based on the reversible photoregulation of DNA hybridization. When non-substituted azobenzene was used, the tweezers were open after UV light irradiation ((330-350 nm, cis form), and closed after visible light irradiation (440-460 nm, trans form). More interestingly, the operation reversed when an azobenzene derivative with a para-isopropyl group was used: UV light irradiation closed the tweezers and visible light irradiation opened them. As compared with the oligonucleotide-fuelled DNA machines, the nanomachines constructed here were “environment-friendly" because no dsDNA waste was produced. Furthermore, the operation can be repeated many times simply by switching the photoirradiation without any decrease of the cycling efficiency.

ＤＮＡを切断するはさみ

西岡英則、浅沼浩之

化学 Vol. 64 page： 70-71 2009

Authorship：Lead author Language：Japanese

遺伝子工学（遺伝子組み換え技術）は、１）遺伝子であるDNAの特定の位置での“裁断”、２）得られたDNA断片の、ベクターと呼ばれる遺伝子の“運び屋”への“縫製”、そして３）ベクターの細胞内への導入（形質転換）、という3つの基本技術に基づいている。この中でも１）のDNAを“裁断”する “はさみ”＝制限酵素　の発見が、現在の遺伝子工学を可能にしたと言っても過言ではない。この天然由来の“はさみ”に相当する制限酵素には様々な種類が存在し、その多くは4～6塩基程度の塩基配列を認識してDNAを切断する。したがって数千塩基程度の短い遺伝子ならば、天然の制限酵素を用いることで特定の１～２箇所を切断することが可能である。しかしながら、高等生物のような巨大なゲノムDNAに対して利用すると非常に多くの断片が生じてしまう。例えば、30億塩基を持つヒトゲノムに対して6塩基しか認識しない制限酵素を利用すると、おおよそ73万 (= 30億÷46)もの断片が生じてしまうことになる。すなわち、天然の制限酵素では、高等生物のDNAを配列特異的に１箇所で切断することは不可能である。このような問題点を克服するために近年、認識配列に制限が無い人工的な “はさみ”＝「人工制限酵素」に関する様々な研究が活発に行われている。ここでは、DNAを配列特異的に切断する人工制限酵素について紹介する。

*Rational Design of Functional DNA with a Non-Ribose Acyclic Scaffold Invited Reviewed

H. Kashida, X.G. Liang, H. Asanuma

Current Organic Chemistry Vol. 13 ( 11 ) page： 1065-1084 2009

Authorship：Lead author Language：English

The growing field of DNA technology requires new modified DNAs that can perform advanced functions. No matter how we optimize the length and sequence of DNA using only the four naturally occurring nucleotides, potential performance is limited. In this review, we describe a facile and effective method of rationally designing new functional DNA by focusing on acyclic scaffolds, especially threoninols, which are utilized to incorporate functional molecules into
DNA. Wedge-type insertion of a functional molecule with a planar structure of proper size in D-threoninol to DNA does
not destabilize the duplex, although the backbone structure is changed. Rather, intercalation offsets such distortions and significantly raises the melting temperature of the DNA duplex. Based on the wedge-type insertion, photoresponsive DNA (tethering azobenzenes) and fluorescent probes that can detect single nucleotide polymorphisms (SNPs) and insertion/
deletion (indel) polymorphisms have been designed. Furthermore, a variety of molecular clusters of dyes have also been prepared from acyclic scaffolds tethering dyes.

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Papers - ASANUMA, Hiroyuki