Faculty Profiles - ASANUMA, Hiroyuki

Papers - ASANUMA, Hiroyuki

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Glucuronidase-assisted transglycosylation for the synthesis of highly functional disaccharides: β-D-Glucuronyl 6-O-sulfo-β-D-gluco- and -β-D-Galactopyranosides Reviewed

Nagatsuka T.; Uzawa H.; Asanuma H., Nishida, Y.

J. Carbohydrate Chem. Vol. 28 page： 94-106 2009

Language：English Publishing type：Research paper (scientific journal)

The substrate specificity of snail, limpet, and bovine glucuronidases was examined by using p-nitrophenyl glucuronide and p-nitrophenyl 6-O-sulfo-D-glycopyranosides as the glycosyl donor and acceptors, respectively. When the donor was treated with these enzymes in the absence of the acceptors, beta(1-3) glucuronyl disaccharides were obtained as the major products together with beta(1-2) isomers as the result of an enzymatic "self-transglycosylation"reaction.

Modulation of pKa of Brooker's Merocyanine by DNA Hybridization Reviewed

Kashida, H.; Sano, K.; Hara, Y.; Asanuma, H.

Bioconjugate Chem. Vol. 20 page： 258-265 2009

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

Brooker'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 nm 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 anti-parallel 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 anti-parallel 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.

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

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

Nucleic Acids Res. Symp. Ser. Vol. 52 page： 697-698 2008.9

Authorship：Lead author Language：English

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 coherent hetero clusters with threoninol scaffold.

Fujii, T.; Kashida, H.; Asanuma, H.

Nucleic Acids Symp. Ser. Vol. 52 page： 699-700 2008.9

Authorship：Lead author Language：English

New hetero aggregates where different dyes stacked alternately were prepared by hybridizing two DNAs, each of which tethered a different dye in the centre of strand. Spectral changes due to exciton coupling between different kinds of dyes were observed. Especially, hetero aggregates of Methyl Red and 2-nitro-4'-dimethylaminoazobenzene showed substantial narrowing of the band, demonstrating coherent coupling occurred in these aggregates.

Incorporation of cationic dyes into DNA for distinct stabilization of duplex

Kashida, H.; Itoh, H.; Fujii, T.; Asanuma, H.

Nucleic Acids Symp. Ser. Vol. 2008 page： 701-702 2008.9

Construction of a photo-switchable gene for turning on and off gene expression with light irradiation

Liang, X.G.; Fujioka, K.; Tsuda, Y.; Wakuda, R.; Asanuma, H.

Nucleic Acids Res. Symp. Ser. Vol. 52 page： 19-20 2008.9

Authorship：Lead author Language：English

A photoresponsive GFP gene was constructed by attaching a T7 promoter that involves two azobenzene moieties as the photoswitch. The azobenzene moieties tethered on D-threoninol were inserted precisely into the sequence of T7 promoter at two positions in the non-template strand. By using azobenzene-tethered DNA as a primer, azobenzene was attached to GFP gene after PCR amplification. However, a single-stranded overhang involving azobenzene was formed because primer extension stopped at the position of azobenzene moiety. Interestingly we found that oligonucleotide complementary to the overhang could be ligated by T4 DNA ligase at the stopped position, and the intact photoresponsive T7 promoter was attached onto GFP gene. Furthermore, the in vitro expression of the constructed photoresponsive GFP gene was successfully switched on and off with light irradiation.

A DNA Nanomachine Powered by Light Irradiation Reviewed

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

ChemBioChem Vol. 9 ( 5 ) page： 702-705 2008.5

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

The photoresponsive DNA tweezers as a nanomachine fuelled with photons were constructed by using azobenzene-modified DNA. The tweezers were photoswitched to be open with the UV light irradiation (335-345 nm) and to be closed with the visible light irradiation (445-455 nm) without further adding oligonucleotides as the fuel.

Postsynthetic modification of DNA via threoninol on a solid support by means of allylic protection Reviewed

Hiroyuki Asanuma, Yuichi Hara, Akira Noguchi, Kanae Sano, Hiromu Kashida

Tetrahedron Letters Vol. 49 page： 5144-5146 2008

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

We have developed a facile but versatile method to introduce functional molecules into DNA on a CPG support. Threoninol, whose amino group was protected with an (allyloxy)carbonyl (Alloc) group, was introduced into DNA via the corresponding phosphoramidite monomer. After selective deprotection of the Alloc group by treatment with palladium(0), a dye with a carboxyl group could be introduced into the DNA on the CPG support through an amide bond.

Threoninol as a Scaffold of Dyes (Threoninol-nucleotide) and Their Stable Interstrand Clustering in Duplexes Reviewed

Hiromu Kashida, Taiga Fujii, Hiroyuki Asanuma

Org. Biomol. Chem. Vol. 6 ( 16 ) page： 2892 - 2899 2008

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

Functional molecules such as dyes (Methyl Red, azobenzene, and Naphthyl Red) were tethered on D-threoninol as base surrogates (threoninol-nucleotide), which were consecutively incorporated at the center of natural oligodeoxyribonucleotides (ODNs). Hybridization of two ODNs involving threoninol-nucleotides allowed interstrand clustering of the dyes on D-threoninol and greatly stabilized the duplex. When two complementary ODNs, both of which had tethered Methyl Reds on consecutive D-threoninols, were hybridized, the melting temperature increased proportionally to the number of Methyl Reds, due to stacking interactions. Clustering of Methyl Reds induced both hypsochromicity and narrowing of the band, demonstrating that Methyl Reds were axially stacked relative to each other (H-aggregation). Since hybridization lowered the intensity of circular dichroism peaks at the p-p* transition region of Methyl Red (300 nm – 500 nm), clustered Methyl Reds were scarcely wound in the duplex. Alternate hetero dye clusters could also be prepared only by hybridization of two ODNs with different threoninol-nucleotides, such as Methyl Red / azobenzene and Methyl Red / Naphthyl Red combinations. A combination of Methyl Red and azobenzene induced bathochromic shift and broadening of the band at the Methyl Red region due to the disturbance of exciton interaction among Methyl Reds. But interestingly, the Methyl Red and Naphthyl Red combination induced merging of each absorption band to give a single sharp band, indicating that exciton interaction occurred among the different dyes. Thus, D-threoninol can be a versatile scaffold for introducing functional molecules into DNA for their ordered clustering.

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

Xingguo Liang, Nobutaka Takenaka, Hidenori Nishioka, Hiroyuki Asanuma

Chem. Asian J. Vol. 9 page： 702-705 2008

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

A new photoswitch of DNA hybridization involving para-substituted azobenzene (such as isopropyl or tert-butyl substituted one) on L-threoninol as a linker was synthesized. When visible light was irradiated to the modified DNA, the duplex was dissociated due to the destabilization effect of the bulky substituent on trans-azobenzene. In contrast, trans-to-cis isomerization (UV light irradiation) facilitated the duplex formation. The direction of this photoswitching mode was entirely reversed as compared with the previous one carrying 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 due to the formation of more stable duplex in cis-form.

Diastereomer Separation of Azobenzene-Tethered Oligodeoxyribonucleotides and Determination of Their Absolute Configurations by Enzymatic Digestion. Reviewed

Xingguo Liang, Makoto Komiyama, Hiroyuki Asanuma

Nucleosides, Nucleotides & Nucleic Acids Vol. 27 page： 332-350 2008

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

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 photo-regulation 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.

Preparation of "comb-type" hetero aggregates by DNA-dye conjugation

Taiga Fujii, Hiromu Kashida, Hiroyuki Asanuma

Nucleic Acids Symp. Ser. Vol. 51 page： 277-278 2007.11

Authorship：Lead author Language：English

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.

Photoregulation of DNA hybridization by introducing an azobenzene: Molecular design for more stabilization of DNA duplex with cis-azobenzene than with its trans-form.

Xingguo Liang, Nobutaka Takenaka, Hidenori Nishioka, Hiroyuki Asanuma

Nucleic Acids Symp. Ser. Vol. 51 page： 169-170 2007.11

Authorship：Lead author Language：English

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-iPrAzo) was introduced into DNA via a L-thereoninol linker, the photoswitching direction was completely reversed: the duplex involving non-planar cis-p-iPrAzo was much more stable than that involving planar trans-form.

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

Xingguo Liang, Tomohiro Kato, Hiroyuki Asanuma

Nucleic Acids Symp. Ser. Vol. 51 page： 351-352 2007.11

Authorship：Lead author Language：English

DNA was synthesized in the absence of DNA or RNA as template (or primer) from dNTPs at relatively low temperatures (25~50oC) by thermophilic Vent DNA polymerase whose proper reaction temperature for primer extension is 70~80oC. Unexpectedly, the ab initio DNA synthesis was even more efficient at 50oC as compared with that at 70oC. 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 intio 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.

Effective photoregulation of gene expression by photoresponsive T7 promoter

Xingguo Liang, Ryuji Wakuda, Yuichiro Tsuda, Hidenori Nishioka, Hiroyuki Asanuma

Nucleic Acids Symp. Ser. Vol. 51 page： 349-350 2007.11

Authorship：Lead author Language：English

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 Km 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 photoregulatoryion 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.

Development of photoresponsive RNA towards photoswitching of RNA functions

Hiroshi Ito, Hidenori Nishioka, Xingguo Liang, Hiroyuki Asanuma

Nucleic Acids Symp. Ser. Vol. 51 page： 171-172 2007.11

Authorship：Lead author Language：English

By introducing azobenzene into RNA via a D-threoninol linker, the photoresponsive RNA was developed for the photoswitching of RNA hybridization. The Tm measurements showed that RNA/RNA duplex formation and dissociation could be efficiently photoregulated. The difference in Tm between trans- and cis-form (DTm) was as large as 9~12oC when azobenzene was tethered at the central position of a 10-nt-long RNA. Interestingly, photoregulation ability of the azobenzene introduced in RNA was even greater than that in DNA for photoswitching the corresponding duplex formation. The constructed photoresponsive RNA is promising to be applied for the photoregulation of RNA functions such as Ribozyme activity, RNAi, pre-mRNA editing, and aptamer complex formation.

Design of peptide nucleic acid by introduction of carbohydrate on lysine linker.

Akira Noguchi, Hiroyuki Asanuma

Vol. 51 page： 261-262 2007.11

Authorship：Lead author Language：English

We synthesized “cartridge" type monomer involving mannose unit on lysine as a linker and additionally in-serted it to peptide nucleic acid (PNA) oligomer. Ad-vantage of this procedure is that functional molecule could be easily introduced into anywhere position of PNA without sacrificing base-pairs, although such in-sertion lowered stability of the duplex with DNA.

Development of high-sensitive DNA probe by using perylene

Hiromu Kashida, Tomohiko Takatsu, Hiroyuki Asansuma

Nucleic Acids Symp. Ser Vol. 51 page： 279-280 2007.11

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

Hiroyuki Asanuma, Xingguo Liang, Hidenori Nishioka, Daijiro Matsunaga, Minghe Liu, Makoto Komiyama

Nature Protocols Vol. 2 ( 1 ) page： 203-212 2007

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

A phosphoramidite monomer bearing an azobenzene is synthesized from D-threoninol. By 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 DNA duplex can be reversibly photo-regulated by cis-trans isomerization of the azobenzene. When the azobenzene takes trans-form, a stable duplex is formed. Upon isomerization of the trans-azobenzene to its cis-form by UV-light irradiation (300 nm < l < 400 nm), the duplex can be dissociated to two strands. The duplex is re-formed on photo-induced cis→trans isomerization (l > 400 nm). By introducing azobenzenes into T7 promoter at specific positions, transcription by T7-RNA polymerase is also efficiently and reversibly photo-regulated. The reversible regulation can be repeated many times without causing damage to DNA or the azobenzene moiety. All these procedures take about 10 days to complete.

Detection of genetic polymorphisms with high sensitivity by DNA-perylene conjugate Reviewed

Hiromu Kashida, Tomohiko Takatsu, Hiroyuki Asanuma

Tetrahedron Letters Vol. 48 page： 6759-6762 2007

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

Modified oligodeoxyribonucleotides (ODNs) involving two perylene moieties are synthesized. By using this ODN, one-base deletion can easily be distinguished with high sensitivity. In addition, emission color of the solution greatly changed so that the detection was possible even by naked eyes.

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Papers - ASANUMA, Hiroyuki