Faculty Profiles - ASANUMA, Hiroyuki

Papers - ASANUMA, Hiroyuki

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Design of a functional nanomaterial with recognition ability for constructing light-driven nanodevices

Liang X., Mochizuki T., Fujii T., Kashida H., Asanuma H.

Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) Vol. 6518 LNCS page： 112 - 122 2011.2

Language：Japanese Publisher：Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)

An artificial macromolecule (foldamer) was designed as a novel nanomaterial with the backbone of phosphodiester and the side chain of functional molecules and nucleobases. The functional molecules tethered on D-threoninol and the nucleosides on D-ribose can be lined up with any sequence and ratio by using standard phosphoramidite chemistry. The nucleobases that form Watson-Crick base pairs provide the sequence recognition which is required for constructing complicate nanostructures. The multiple functional molecules give applicable and advanced functions such as photoresponsiveness when azobenzenes were used. Unexpectedly, a stable double helix was formed even in the case that the ratio of azobenzene molecules and base pairs was as high as 2:1. More interestingly, this artificial duplex showed high sequence specificity: the stability decreased greatly when a mismatched base pair was present. Furthermore, the formation and dissociation of the constructed artificial duplex were reversibly and completely modulated with light irradiation. By using this new nanomaterial, a variety of functional nanostructures and nanodevices are promising to be designed. © 2011 Springer-Verlag.

DOI： 10.1007/978-3-642-18305-8_11

A light-driven DNA nanomachine for the efficient photoswitching of RNA digestion

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

Angewandte Chemie - International Edition Vol. 49 ( 12 ) page： 2167 - 2170 2010.3

Language：Japanese Publisher：Angewandte Chemie - International Edition

"Chemical Equation Presented" Photons as fuel: A photoresponsive DNA enzyme was constructed that can work at the single-molecule level. Complete ONOFF photoswitching of RNA digestion was realized by photoregulating the topological structure of a DNAzyme/RNA complex. The key components of the photoswitch are azobenzene units. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/anie.200907082

Photoregulation of DNA transcription by using photoresponsive T7 promoters and clarification of its mechanism

Liang X., Wakuda R., Fujioka K., Asanuma H.

FEBS Journal Vol. 277 ( 6 ) page： 1551 - 1561 2010.3

Language：Japanese Publisher：FEBS Journal

With the use of photoresponsive T7 promoters tethering two 2′-methylazobenzenes 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→trans thermal stability between cis-nonmodified azobenzene and cis-2′,6′- dimethylazobenzene, 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 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. © 2010 FEBS.

DOI： 10.1111/j.1742-4658.2010.07583.x

Molecular imprinting of cyclodextrin to physiologically active oligopeptides in water

Song S., Shirasaka K., Hirokawa Y., Asanuma H., Wada T., Sumaoka J., Komiyama M.

Supramolecular Chemistry Vol. 22 ( 3 ) page： 149 - 155 2010.3

Language：Japanese Publisher：Supramolecular Chemistry

β-Cyclodextrin (β-CyD)-based polymeric receptors for γ-endorphin (γ-endor, an opioid heptadecapeptide) were prepared using the molecular imprinting method. When mono-3-(N-acrylamido)-3-deoxy-β-CyD bearing a vinyl group in the secondary hydroxyl side of the cavity of β-CyD was polymerised in water in the presence of γ-endor, the binding activity of the β-CyD polymer to this peptide in water was enormously promoted by the imprinting. By contrast, the bindings towards methionine-enkephalin (N-terminal pentapeptide of γ-endor) and its homologue leucine-enkephalin were suppressed. Thus, the binding of γ-endor by the imprinted polymer was highly selective. The imprinting towards γ-endor was also successful with the use of the β-CyD monomer bearing a vinyl group in the primary hydroxyl side of the cavity, although the recognition was less strict. Various factors affecting the imprinting efficiency (kinds of β-CyD vinyl monomer and template, as well as the pH of imprinting mixture) are discussed. © 2010 Taylor & Francis.

DOI： 10.1080/10610270902980622

An efficient fluorescence resonance energy transfer (FRET) between Pyrene and Perylene assembled in a DNA duplex and its potential for discriminating single-base changes

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

Chemistry - A European Journal Vol. 16 ( 8 ) page： 2479 - 2486 2010.2

Language：Japanese Publisher：Chemistry - A European Journal

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 through d-threoninol and natural base pairs were inserted between the dyes. Accordingly, donors and acceptors could be accumulated inside the DNA without forming an 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 at which pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high apparent FRET efficiency (Fφ1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Fçrster radius (R0) of the donor pyrene. Next, this FRET system was used to enlarge the Stokes' shift of the DNA probe, which can discriminate a one-base deletion mutant from wild type with a model system by incorporation of multiple donors into DNA. Two perylene moieties were tethered to the DNA on both sides of the intervening base, and two pyrenes were further inserted in the vicinity of the perylenes as an antenna. Hybridization of this FRET probe with a fully matched DNA allowed monomer emission of perylene when the pyrenes were excited. In contrast, excimer emission was generated by hybridization with a one-base deletion mutant. Thus, the apparent Stokes' shift was enhanced without loss of efficiency in the detection of the deletion mutant. © 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.

DOI： 10.1002/chem.200902078

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

Nishioka H., Liang X., Asanuma H.

Chemistry - A European Journal Vol. 16 ( 7 ) page： 2054 - 2062 2010.2

Language：Japanese Publisher：Chemistry - A European 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 T m (ΔTm) 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 ΔTm, 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 ΔTm. 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 cis2,6-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 eis form was attributed to the retardation of the inversion process due to steric hindrance between lone pair electrons of the π orbital of the nitrogen atom and the methyl group on the distal benzene ring. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/chem.200902789

Femtosecond photoisomerization study on azobenzene-derivative bound by DNA

Chen T., Igarashi K., Yamaguchi A., Nakagawa N., Yamane K., Fujii T., Asanuma H., Yamashita M.

Optics InfoBase Conference Papers 2010

Analysis of coherent heteroclustering of different dyes by Use of threoninol nucleotides for comparison with the molecular exciton theory

Fujii T., Kashida H., Asanuma H.

Chemistry - A European Journal Vol. 15 ( 39 ) page： 10092 - 10102 2009.10

Language：Japanese Publisher：Chemistry - A European 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 oligodeoxyribonucleotides (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. The spectroscopic behavior of these heterodimers was systematically examined as a function of the difference in the wavelength of the dye absorption maxima (Δλmax). 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 λmax at 336 and 480 nm, respectively, in the single strand (Δλmax= 144nm), 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, which had a much smaller Δλmax (82 nm; λmax = 398 and 480 nm in the single-strand, respectively). With a combination of 4'-dimethyl-amino-2- nitroazobenzene and Methyl Red, which had an even smaller Δλ 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, which has been mainly applied to the spectral behavior of H- and/or Jaggregates composed of homo dyes. However, the bathochromic band shifts observed at shorter wavelengths did not agree with the hypsochromism predicted by the theory. Thus, these data experimentally verify the molecular exciton theory of heterodimerization. This coherent coupling among the heterodimers could also partly explain the bathochromicity and hypochromicity that were observed when the dyes were intercalated into the duplex. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/chem.200900962

In-stem molecular beacon containing a pseudo base pair of threoninol nucleotides for the removal of background emission

Kashida H., Takatsu T., Fujii T., Sekiguchi K., Liang X., Niwa K., Takase T., Yoshida Y., Asanuma H.

Angewandte Chemie - International Edition Vol. 48 ( 38 ) page： 7044 - 7047 2009.9

Language：Japanese Publisher：Angewandte Chemie - International Edition

Off means off: An in-stem molecular beacon in which Dthreoninol units tether perylene and anthraquinone in the stem region effectively detected target sequences and was able to discriminate a one-base-deletion mutant from the wild-type (full-match) sequence without background emission (see picture). © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/anie.200902367

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

Liang X., Mochizuki T., Asanuma H.

Small Vol. 5 ( 15 ) page： 1761 - 1768 2009.8

Language：Japanese Publisher：Small

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 whereNandXrepresent 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. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/smll.200900223

Positively charged base surrogate for highly stable "base pairing" through electrostatic and stacking interactions

Kashida H., Ito H., Fujii T., Hayashi T., Asanuma H.

Journal of the American Chemical Society Vol. 131 ( 29 ) page： 9928 - 9930 2009.7

Language：Japanese Publisher：Journal of the American Chemical Society

(Figure Presented) "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. © 2009 American Chemical Society.

DOI： 10.1021/ja9013002

Modulation of pK<inf>a</inf> of Brooker's merocyanine by DNA hybridization

Kashida H., Sano K., Kara Y., Asanuma H.

Bioconjugate Chemistry Vol. 20 ( 2 ) page： 258 - 265 2009.2

Language：Japanese Publisher：Bioconjugate Chemistry

Brocker's merocyanine (BM), which changes its emission and absorption maxima upon protonation, was introduced into oligodeoxyribonucleotide (ODN) via D-threoninol by postsynthetic modification on a CPG (controlled-pore glass) support. The pKa of BM in the modified ODN increased from 9.5 to 10.1 upon hybridization. As a result, absorption maxima shifted from 492 to 432 nm at pH 10.0 by the presence of its complementary strand. This spectral shift was sufficiently large so that DNA hybridization could easily be discriminated even by the naked eye; the color of the solution changed from orange to yellow upon hybridization. In addition, the fluorescence emission was strongly quenched upon hybridization, demonstrating that this probe can also detect the target DNA by the fluorescence change. Ratiometric detection of hybridization was also possible by simultaneous excitation of both protonated and deprotonated BMs. Furthermore, we could also modulate its pKa by the antiparallel stacking of two BM molecules in the duplex; the pKa of BM decreased from 10.1 to 9.7 by the stacking of two BMs in an antiparallel manner. Thus, control of the microenvironment around the BM molecule allowed modulation of its pKa, which is applicable to the sequence-specific recognition of target DNA. © 2009 American Chemical Society.

DOI： 10.1021/bc800335h

Rational design of functional DNA with a Non-Ribose acyclic scaffold

Kashida H., Liang X., Asanuma H.

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

Language：Japanese Publisher：Current Organic Chemistry

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. © 2009 Bentham Science Publishers Ltd.

DOI： 10.2174/138527209788680736

名古屋コンファレンス報告

浅沼浩之, 早川芳宏

化学と工業 = Chemistry and chemical industry Vol. 60 ( 4 ) page： 456 2007.4

2′,6′-Dimethylazobenzene as an efficient and thermo-stable photo-regulator for the photoregulation of DNA hybridization

Nishioka H., Liang X., Kashida H., Asanuma H.

Chemical Communications ( 42 ) page： 4354 - 4356 2007

Unexpected efficient ab initio DNA synthesis at low temperature by using thermophilic DNA polymerase.

Liang X., Kato T., Asanuma H.

Nucleic acids symposium series (2004) ( 51 ) page： 351 - 352 2007

Language：English Publisher：Nucleic acids symposium series (2004)

DNA was synthesized in the absence of DNA or RNA as template (or primer) from dNTPs at relatively low temperatures (25 approximately 50 degrees C) by thermophilic Vent DNA polymerase whose proper reaction temperature for primer extension is 70 approximately 80 degrees C. Unexpectedly, the ab initio DNA synthesis was even more efficient at 50 degrees C as compared with that at 70 degrees C. Interestingly, the ab initio DNA synthesis by Vent (exo), a mutant version of Vent DNA polymerase lacking of 3' --> 5' exonuclease activity, became much less efficient, and it could only carry out ab initio DNA synthesis after a long incubation time. This remarkable difference between Vent and Vent (exo) indicates that the exonuclease activity domain of Vent may play an important role at the initiation step of ab initio DNA synthesis.

DOI： 10.1093/nass/nrm176

Effective photoregulation of DNA hybridization by the introduction methyl group into azobenzene

Nishioka H., Kashida H., Komiyama M., Asanuma H.

Polymer Preprints, Japan Vol. 55 ( 1 ) 2006.10

Language：Japanese Publisher：Polymer Preprints, Japan

We have synthesized azobenzene-tethered DNA and have successfully photoregulated various DNA functions. In the present study, we synthesized various azobenzenes substituted with methyl group for still more effective photoregulation, as shown in Fig. 1. The melting temperatures of 1a/S 0 duplex are listed in Table 1. When an azobenzene took trans-form, mono-substituted one at ortho position exhibited larger Tm compared with other mono-substituted ones. In contrast, Tm of ortho-substituted azobenzene was the smallest in Tm-form. As a result, change of T m (ΔTm) induced by trans-cis isomerization was as large as 10.6°C, which was 4.9°C higher than previous non-substituted azobenzene (Azo: 5.7°C). Quite interestingly, di-substituted azobenzene at both ortho positions (2,6-Azo) exhibited even larger ΔTm (14.6°C). Thus, we succeeded in the efficient photoregulation of DNA hybridization by the modification of azobenzene at ortho position.

Preparation of active DNA enzymes by introduction of intercalator

Hayashi H., Zhao J., Komiyama M., Liang X., Asanuma H.

Polymer Preprints, Japan Vol. 55 ( 1 ) 2006.10

Language：Japanese Publisher：Polymer Preprints, Japan

The RNA cleaving 10-23 DNA enzyme, which is expected to be used as an antisense agent to suppress gene activity, shows dependence of activity on the sequence at the active site. We already showed that the cleavage efficiency of 10-23 DNA enzyme with lower activity was improved by attaching an azobenzene residue close to the active site. In this study, we introduced several planar molecules instead of azobenzene as intercalators and checked their activation (Fig. 1). We found that structures of introduced intercalators and linkers greatly influenced the cleavage activity. When anthraquinone was used as the intercalator, catalytic activation was increased more than six folds in comparison with the native DNA enzyme (Table 1). Our finding is helpful for understanding the mechanism of cleavage by DNA enzymes.

Synthesis of peptide nucleic acid conjugated with carbohydrate

Noguchi A., Yamada Y., Nishida Y., Asanuma H.

Polymer Preprints, Japan Vol. 55 ( 1 ) 2006.10

Language：Japanese Publisher：Polymer Preprints, Japan

Peptide nucleic acid (PNA) is composed of non-charged N-2-aminoethylglycine as a scaffold, and thus it strongly hybridizes with natural DNA. But due to its high hydrophobic property, large PNA oligomers prefer to make aggregates rather than form duplex with DNA. Therefore, incorporation of functional molecules such as intercalators is difficult because such modification should raise the hydrophobicity of PNA. In order to modify the PNA without increasing its hydrophobicity, we designed a new monomer involving carbohydrate for the introduction of PNA oligomer. Here, we successfully synthesized a lysine monomer tethering mannose unit according to the Scheme 1. In the present paper, effect of mannose on the stability of PNA/DNA duplex is examined.

Synthesis of fluorescent DNA probe by use of dye polarization

Sano K., Tanaka M., Kashida H., Komiyama M., Asanuma H.

Polymer Preprints, Japan Vol. 55 ( 1 ) 2006.10

Language：Japanese Publisher：Polymer Preprints, Japan

DNA duplex involves hydrophobic inside in which water molecules are excluded, and its exterior is surrounded with the anions of phosphodiester linkages. Since the inside is not shielded by water molecules, strong electric field is formed by the phosphodiester anion and thus the molecules bound to the duplex should be easily polarized. In the present study, we tethered Dansyl moiety as a fluorophore through amide bond to DNA (Dco in Scheme 1), and fluorescence change by duplex formation was investigated. Single-stranded S showed fluorescence emission at around 550 nm (Blue line in Fig.1). When it was hybridized with native S0, small but distinct decrease in the fluorescence by the phosphodiester anion was observed (Red line). On the contrary, hybridization with M56 (Green line), in which two phosphodiesters nearest to Dansyl moiety are replaced with non-charged methyl phosphonate, slightly increased the fluorescence.

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