Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.1021/acs.joc.2c01911

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

DOI： DOI: 10.1126/scirobotics.abm0677

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

DOI： 10.1039/d1cc05868a

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Society of Synthetic Organic Chemistry, Japan  

<p>Natural base pairs stabilize nucleic acid duplexes via hydrogen bonding and stacking interactions, and efforts have been made to identify artificial base pairs that stabilize duplexes through different interactions. Herein, we describe our attempts to prepare pseudo base pairs that exhibit high stability and orthogonality. Pairs of azobenzene derivatives showed unexpectedly high stability and orthogonality due to intermolecular stacking interactions. Pairs of cationic molecules showed even higher stability than natural G-C pairs through both electrostatic and stacking interactions. Duplexes can be crosslinked via [2+2] photocycloaddition between stilbene derivatives, and pairs of non-planar cyclohexane derivatives can stabilize duplexes by hydrophobic interactions rather than stacking interactions. Hetero-selective pairing can be achieved using electron donor-acceptor interactions. These pseudo base pairs would work well as building blocks in nanotechnology and biotechnology due to their unique functionalities.</p>

DOI： 10.5059/yukigoseikyokaishi.79.1013

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CiNii Research

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemistry - A European Journal  

We report a new Förster resonance energy transfer (FRET) system for structural analyses of DNA duplexes using perylene and Cy3 as donor and acceptor, respectively, linked at the termini of a DNA duplex via D-threoninol. Experimentally obtained FRET efficiencies were in good agreement with theoretical values calculated based on canonical B-form DNA. Due to the relatively long Förster radius, this system can be used to analyze large DNA structures, and duplexes containing photo-reactive molecules can be analyzed since perylene can be excited with visible light. The system was used to analyze a DNA duplex containing stilbene, demonstrating that in the region of the stilbene cluster the duplex adopts a ladder-like structure rather than helical one. Upon photodimerization between stilbene residues, FRET efficiencies indicated the reaction does not disturb DNA duplex. This FRET system will be useful for analysis of photoreactions of nucleobases as well as a wide range of nucleic acid structures.

DOI： 10.1002/chem.202101738

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PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ChemBioChem  

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.

DOI： 10.1002/cbic.202100184

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Language：Japanese  

DOI： 10.1002/cpz1.106

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PubMed

Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/chem.202003528

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PubMed

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Sensors  

microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cy-tosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the sub-sequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer-or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demon-strated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.

DOI： 10.3390/s21051785

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/d0sc05245k

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PubMed

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

DOI： 10.1021/acsami.0c22314

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PubMed

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

DOI： 10.1002/cptc.202000199

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemistry - A European Journal  

Herein is reported a circularly polarized luminescent (CPL) probe that can respond to the chirality of nucleic acids. An achiral nanostructure was prepared by the hybridization of symmetric serinol nucleic acid (SNA) containing pyrene-modified residues. When chiral oligomers that were complementary to the SNA were added, they induced helicity into the SNA nanowire. Efficient circular dichroism (CD) signal amplification was observed when pyrene was attached to uracil bases through a rigid alkynyl linker. Both CPL and CD signals were observed; they depended on the chirality of the added acyclic threoninol nucleic acid (aTNA) oligomer. This system can be used to convert the chirality of chiral biomolecules into chiroptical signals.

DOI： 10.1002/chem.202102333

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PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Society for the Study of the Origin and Evolution of Life Japan  

<p>  We have developed artificial <i>acyclic</i> nucleic acids composed of serinol derivatives as scaffolds. Recently, template-directed synthesis of <i>acyclic</i> <span style="font-variant: small-caps;">L</span>-threoninol nucleic acid (<span style="font-variant: small-caps;">L</span>-<i>α</i>TNA) could be achieved non-enzymatically via chemical ligation mediated by <i>N</i>-cyanoimidazole in the presence of Mn²⁺ ion. A pseudo primer extension reaction like natural polymerase was also attained non-enzymatically using a pool of random <span style="font-variant: small-caps;">L</span>-<i>α</i>TNA trimers. In this short review, we discuss the possibility of <span style="font-variant: small-caps;">L</span>-<i>α</i>TNA as a candiate of pre-RNA before RNA world by focusing on the evolution of genetic material.</p>

DOI： 10.50968/vivaorigino.49_9

CiNii Research

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s42004-020-00400-2

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

DOI： 10.3390/ijms21155218

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

DOI： 10.1039/d0cc03124k

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PubMed

Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/anie.202004221

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

DOI： 10.1039/d0cc01865a

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

DOI： 10.1002/asia.201901728

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

DOI： 10.1002/cbic.201900498

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

DOI： 10.1021/jacs.9b03267

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

DOI： 10.1002/cptc.201900060

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

DOI： 10.1002/anie.201901272

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

DOI： 10.1039/c8an02520g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

<p>We synthesized <i>N</i>-benzoyl-protected peptide nucleic acid (PNA) monomers, which are robust under the conditions for deprotecting the 9-fluorenylmethoxycarbonyl (Fmoc) group by piperidine but are removable by aqueous ammonia and hence totally compatible with Fmoc-solid phase synthesis. This new invention expands the range of available nucleobase sequences, allowing us to use acid-sensitive PNA oligomers and purine nucleotides (both of which are difficult to use in the conventional methods) in the preparation of PNA-DNA chimeras to avoid the drawbacks of traditional PNAs.</p>

DOI： 10.1246/cl.181048

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CiNii Research

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

DOI： 10.1371/journal.pone.0211505

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

DOI： 10.1039/c8cc09218d

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

DOI： 10.1039/c9ob00946a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

<p>In this account, we demonstrate that DNA duplex is an ideal scaffold for photochemistry, particularly for comparison of photochemical theory with experiments. The well-defined structure of a DNA duplex can be regarded as an aqueous one-dimensional soft crystal composed of a chromophore-like base-pair assembly. When any base pair in the duplex is replaced with a chromophore, orientation, distance, and association number of chromophores can be precisely controlled. We have developed a new methodology for introduction of chromophores into DNA duplexes using <span style="font-variant: small-caps;">d</span>-threoninol. By using the DNA duplex as a scaffold, experiments on exciton interactions of chromophore assemblies can be compared with molecular exciton theory. A fluorescent resonance energy transfer (FRET) system was also constructed by introducing donor pyrene and acceptor perylene into the DNA duplex using <span style="font-variant: small-caps;">d</span>-threoninol monomers. Using this system, we demonstrated orientation-dependent FRET. We found that theories on both exciton interaction and FRET qualitatively coincide with experimental data and revealed the limitation of the point-dipole approximation. We also evaluated the intrinsic quantum yield of photodimerization of stilbene derivatives by suppressing a side reaction. We propose that there is a correlation of quantum yield of photodimerization with the energy gap of HOMO or LUMO, a hypothesis that deserves theoretical investigation.</p>

DOI： 10.1246/bcsj.20180278

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CiNii Research

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

DOI： 10.1038/s42004-018-0093-0

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

DOI： 10.1021/jacs.8b02807

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

DOI： 10.1038/s41467-017-02778-5

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Publisher：株式会社医学書院  

DOI： 10.11477/mf.2425200684

CiNii Research

Language：Japanese   Publisher：Cardiovascular Drugs and Therapy  

Purpose: We evaluated the effects of an alpha-glucosidase inhibitor, voglibose, on cardiovascular events in patients with a previous myocardial infarction (MI) and impaired glucose tolerance (IGT). Methods: This prospective, randomized, open, blinded-endpoint study was conducted in 112 hospitals and clinics in Japan in 3000 subjects with both previous MI and IGT receiving voglibose (0.6 mg/day, n = 424) or no drugs (n = 435) for 2 years. The Data and Safety Monitoring Board (DSMB) recommended discontinuation of the study in June 2012 after an interim analysis when the outcomes of 859 subjects were obtained. The primary endpoint was cardiovascular events including cardiovascular death, nonfatal MI, nonfatal unstable angina, nonfatal stroke, and percutaneous coronary intervention/coronary artery bypass graft. Secondary endpoints included individual components of the primary endpoint in addition to all-cause mortality and hospitalization due to heart failure. Results: The age, ratio of males, and HbA1C were 65 vs. 65 years, 86 vs. 87%, and 5.6 vs. 5.5% in the groups with and without voglibose, respectively. Voglibose improved IGT; however, Kaplan–Meier analysis showed no significant between-group difference with respect to cardiovascular events [12.5% with voglibose vs. 10.1% without voglibose for the primary endpoint (95% confidence interval, 0.82–1.86)]; there were no significant differences in secondary endpoints. Conclusion: Although voglibose effectively treated IGT, no additional benefits for cardiovascular events in patients with previous MI and IGT were observed. Voglibose may not be a contributing therapy to the secondary prevention in patients with MI and IGT. Trial Registration: Clinicaltrials.gov number: NCT00212017.

DOI： 10.1007/s10557-017-6740-3

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

DOI： 10.1039/c7ra06091b

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Language：English   Publisher：公益社団法人 日本化学会  

<p>The authors have developed various kinds of pseudo base pairs using a <span style="font-variant: small-caps;">d</span>-threoninol scaffold. Although the chemical structures of the pseudo base pairs are much different from natural nucleobases, they can mimic supramolecular properties of natural base pairs. Moreover, modified DNA can possess various functions that cannot be achieved by natural nucleic acids, such as fluorescent switchability, photocrosslinking, insulating and emission color change. These pseudo base pairs can be used to prepare various functional nanomaterials. In the present account, we summarize our recent work on pseudo base pairs, focusing on molecular designs and functions.</p>

DOI： 10.1246/bcsj.20160371

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Language：Japanese   Publisher：Chemistry - A European Journal  

Reversible photo-cross-linking of a DNA duplex through the [2+2] photocycloaddition of styrylpyrene is reported. Styrylpyrene moieties on d-threoninol linkers were introduced into complementary positions on DNA strands. Irradiation of the styrylpyrene pair in the duplex with visible light at λ=455 nm induced a [2+2] photocycloaddition between styrylpyrenes that cross-linked the two strands of the duplex. Two diastereomers were formed after [2+2] photocycloaddition as a result of rotation of the styrylpyrene residues. Also, the cycloreversion reaction was induced by UV light at λ=340 nm, which reversibly yielded the uncross-linked strands.

DOI： 10.1002/chem.201602006

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PubMed

Language：Japanese   Publisher：Journal of Controlled Release  

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.

DOI： 10.1016/j.jconrel.2016.02.048

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CiNii Research

Language：Japanese   Publisher：Biomaterials Science  

We report the development of photo-crosslinked siRNA strands modified at each terminus with p-cyanostilbene. The siRNA was nuclease resistant and retained RNAi activity. We further studied the activation mechanism of the covalently-crosslinked siRNA. Interestingly Dicer, which is known to generate siRNA with overhanging 3′ ends from the precursor siRNA, did not cleave the crosslinked siRNA at all. Our results suggest that the activation of the crosslinked siRNAs required cleavage by Argonaute2.

DOI： 10.1039/c5bm00231a

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Language：Japanese   Publisher：ChemBioChem  

The 3 D structure of Cas9 bound with sgRNA was solved by X-ray crystal-structure analysis. The conformation change in SpyCas9 upon binding to sgRNA changes Cas9 to target-DNA-recognition mode in which seed segments in sgRNA are pre-ordered in an A-type conformation.

DOI： 10.1002/cbic.201500424

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PubMed

DOI： 10.11477/mf.2425200121

CiNii Research

Language：Japanese   Publisher：Lecture Notes in Electrical Engineering  

DNA detection is widely used for gene analysis. However, false-positive results are difficult to be avoided due to the non-specific amplification. Here, we described a novel RCA approach to improve the specificity. With the help of TspR I (endonuclease), DNA was cleaved into smaller duplex fragments, including the target DNA fragment, with 9 nt sticky ends. A duplex adaptor with two sticky ends which were complementary to that of the target fragment was designed. After hybridization of the adaptor with the target fragments, T4 DNA ligase was used to ligate them to form a circular ds DNA with a gap. Then RCA was carried out from the free 3′-end of the open strand by Phi29 DNA polymerase with two primers which were complementary to the target sequence. Different from the traditional padlock-RCA, SC-RCA showed excellent specificity by amplifying the specific DNA target other than the added probe. © Springer-Verlag Berlin Heidelberg 2014.

DOI： 10.1007/978-3-642-37925-3_154

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Language：Japanese   Publisher：Kobunshi  

We synthesized a unique nucleic acid analogue, Serinol Nucleic Acid (SNA), from a serinol scaffold tethering natural nucleobases through an amide bond. The SNA oligomer that is fully synthesized from four chiral SNA monomers changes its chirality by the sequence design, not by the inherent chirality of monomers: The SNA oligomer with an asymmetric sequence is chiral whereas the symmetric one is achiral. The chirality of the SNA oligomer can be inverted by reversing its sequence, i.e., two enantiomers of SNA oligomers can be synthesized from four monomers with the identical chirality. Interestingly, SNA formed a remarkably stable duplex with a complementary SNA in an antiparallel manner with its helicity depending on the sequence, which is much more stable than the corresponding DNA/DNA or RNA/RNA duplex. More interestingly, SNA also formed a stable duplex both with DNA and RNA, indicating high potential for antisense agents. These unique properties of SNA might provide an insight for why D-ribose was selected as a scaffold for natural nucleic acids. © 2013 The Society of Polymer Science, Japan.

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Language：Japanese   Publisher：Chemical Science  

The effects of modification of the phosphodiester backbone or the guanine bases on G-quadruplex formation have been widely investigated. Only a few studies have investigated the effects of deoxyribose or ‘sugar’ modifications on G-quadruplex structure. Here, we evaluated the structural, thermodynamic, and kinetic properties of the parallel quadruplexes formed by the sequence d(TGGGGT) in which each guanine base was substituted, one at a time, with acyclic threoninol nucleic acid (aTNA). We found that all sequences were able to form G-quadruplexes; however, the presence of aTNA resulted in the formation of a mixture of quadruplex structures in some cases. Furthermore, the presence of a single substitution at any position resulted in destabilization of the G-quadruplex relative to that formed by the unmodified sequence. The introduction of the aTNA in terminal quartets was the most detrimental to stability. In addition, kinetic experiments showed that, compared to its unmodified counterpart sequence d(TGGGGT), the substitution of a normal guanine nucleotide by aTNA decelerated quadruplex formation except when the aTNA was at the 5′ most guanine of the sequence. In summary, our studies indicate that the deoxyribose sugar affects the properties of G-quadruplex structures. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3sc50474c

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Language：Japanese   Publisher：Journal of the American Chemical Society  

In this study, we report a photo-cross-linking reaction between p-stilbazole moieties. p-Stilbazoles were introduced into base-paring positions of complementary DNA strands. The [2 + 2] photocycloaddition reaction occurred rapidly upon light irradiation at 340 nm. Consequently, duplex was cross-linked and highly stabilized after 3 min irradiation. The CD spectrum of the cross-linked duplex indicated that the B-form double-helical structure was not severely distorted. NMR analysis revealed only one conformation of the duplex prior to UV irradiation, whereas two diastereomers were detected after the photo-cross-linking reaction. Before UV irradiation, p-stilbazole can adopt two different stacking modes because of rotation around the single bond between the phenyl and vinyl groups; these conformations cannot be discriminated on the NMR time scale due to rapid interconversion. However, photo-cross-linking fixed the conformation and enabled discrimination both by NMR and HPLC. The artificial base pair of p-methylstilbazolium showed almost the same reactivity as p-stilbazole, indicating that positive charge does not affect the reactivity. When a natural nucleobase was present in the complementary strand opposite p-stilbazole, the duplex was significantly destabilized relative to the duplex with paired p-stilbazole moieties and no photoreaction occurred between p-stilbazole and the nucleobase. The p-stilbazole pair has potential as a "third base pair" for nanomaterials due to its high stability and superb orthogonality. © 2013 American Chemical Society.

DOI： 10.1021/ja401835j

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Language：Japanese   Publisher：Chemical Science  

We have developed a new quencher-free stemless linear probe involving multiple perylenes incorporated through d-threoninol; each perylene is separated by intervening natural nucleotides. Without a substrate, the flexible linear probe does not emit fluorescence due to the self-quenching of the weakly interacting fluorophores. Upon hybridization with the target, intercalation of each dye between the base pairs results in emission of strong fluorescence. The maximum signal-background ratio attained was 180, and the response rate was significantly faster than that of a classic hairpin-forming molecular beacon. © 2012 The Royal Society of Chemistry.

DOI： 10.1039/c2sc20732j

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Language：Japanese   Publisher：Chemistry - A European Journal  

We prepared reversed dye clusters by hybridizing two RNA oligomers, each of which tethered dyes (Methyl Red, 4'-methylthioazobenzene, and thiazole orange) on D-threoninols (threoninol nucleotides) at the center of their strands. NMR spectroscopic analyses revealed that two dyes from each strand were axially stacked in an antiparallel manner to each other in the duplex, and were located adjacent to the 3'-side of a natural nucleobase. Interestingly, this positional relationship of the dyes was completely the opposite of that assembled in DNA that we reported previously: dyes in DNA were located adjacent to the 5'-side of a natural nucleobase. This observation was also consistent with the circular dichroism of dimerized dyes in which the Cotton effect of the dyes (i.e., the winding properties of two dyes) was inverted in RNA relative to that in DNA. Further spectroscopic analyses revealed that clustering of the dyes on RNA duplexes induced distinct hypsochromicity and narrowing of the band, thus demonstrating that the dyes were axially stacked (i.e., H-aggregates) even on an A-type helix. On the basis of these results, we also prepared heterodimers of a fluorophore (thiazole orange) and quencher (Methyl Red) in an RNA duplex. Fluorescence from thiazole orange was found to be strongly quenched by Methyl Red due to the excitonic interaction, so that the ratio of fluorescent intensities of the RNA-thiazole orange conjugate with and without its complementary strand carrying a quencher became as high as 27. We believe that these RNA-dye conjugates are potentially useful probes for real-time monitoring of RNA interference (RNAi) mechanisms. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/chem.201201956

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Language：Japanese   Publisher：Biochemistry  

Short DNA sequences, especially those that are repetitive or palindromic, can be used as the seeds for synthesis of long DNA by some DNA polymerases in an unusual manner. Although several elongation mechanisms have been proposed, there is no well-established model that explains highly efficient elongation under isothermal conditions. In the present study, we analyzed the elongation of nonrepetitive sequences with distinct hairpins at each end. These DNAs were elongated efficiently under isothermal conditions by thermophilic Vent (exo -) DNA polymerase, and the products were longer than 10 kb within 10 min of the reaction. A 20-nucleotide DNA with only one hairpin was also elongated. Sequence analysis revealed that the long products are mainly tandem repeats of the short seed sequences. The thermal melting temperatures of the products were much higher than the reaction temperature, indicating that most DNAs form duplexes during the reaction. Accordingly, a terminal hairpin formation and self-priming extension model was proposed in detail, and the efficient elongation was explained. Formation of the hairpin at the 5′ end plays an important role during the elongation. © 2012 American Chemical Society.

DOI： 10.1021/bi3010413

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Language：Japanese   Publisher：Angewandte Chemie - International Edition  

DNA seesaw: Photoswitchable azobenzenecarboxylic acid 1 reversibly photoisomerizes between the trans form and the thermally stable cis form upon irradiation with visible light. A photon-fueled DNA nanodevice that moves like a seesaw in response to irradiation with different wavelengths of light was made by modifying DNA oligonucleotides with a combination of 1 and a conventional azobenzene (see picture). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/anie.201106093

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Language：Japanese   Publisher：Chemical Communications  

In the presence of poly(l-lysine)-graft-dextran, an in-stem molecular beacon involving three perylene–anthraquinone pairs in the stem region had a signal/background ratio of as high as 570. Response speed was also remarkable; equilibrium was attained within 5 minutes after addition of substrate DNA at 20 °C. © 2012 The Royal Society of Chemistry.

DOI： 10.1039/c2cc16812j

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Language：Japanese   Publisher：Chemistry - A European Journal  

Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90 % in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis,. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/chem.201100215

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Language：Japanese   Publisher：Chemistry - A European Journal  

In this study, we propose that three consecutive cationic p-methylstilbazoles tethered on D-threoninols (Z residues) at 5′ termini act as a unique "glue" connecting DNA duplexes by their interstrand cluster formation. Interstrand clustering of p-methylstilbazoles (ZZZ triplets) induces narrowing and hypsochromic shift of bands at 350nm, which can be assigned to the absorption of p-methylstilbazole. However, single-stranded DNA conjugates involving a ZZZ triplet at the 5′ terminus of 8-mer native nucleotides is found not to induce such large spectral changes, which implies that the intrinsic self-assembling property of ZZZ triplets is weak. Interestingly, when this conjugate is hybridized with a complementary 8-mer native oligonucleotide, a remarkable spectral change is observed, indicating the dimerization of a duplex through the interstrand clustering of ZZZ triplets. Dimerization of the duplex is also evidenced by cold-spray ionization mass spectrometry. This interstrand clustering is observed only when a ZZZ triplet is tethered to a 5′ rather than 3′ terminus. Furthermore, the stability of the interstrand cluster increases by increasing the number of nucleobases of the DNA portion, and when mismatched base pairs are incorporated or when a base next to the Z residue is deleted, the stability substantially drops. When we apply the ZZZ triplet to the formation of a nanowire using two complementary DNA conjugates, each of which has a ZZZ triplet at the 5′ termini as overhang, we demonstrate the successful formation of a nanowire by native PAGE analysis. Since native sticky ends that have three nucleotides do not serve as "glue", ZZZ triplets with their unique glue-like properties are prime candidates for constructing DNA-based nanoarchitectures. Stick together! A unique "glue" of cationic p-methylstilbazoles tethered on D-threoninols connects DNA duplexes by intermolecular clustering. When the "glue" is attached to both termini of a DNA duplex, a nanowire is formed (see graphic). Thus, the "glue" can be utilized for constructing DNA-based nanoarchitectures. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/chem.201003059

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PubMed

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

Scopus

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

Scopus

PubMed

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

Scopus

PubMed

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

Scopus

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

Scopus

PubMed

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

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PubMed

Language：Japanese   Publisher：Optics InfoBase Conference Papers  

First observation of femtosecond absorbance change in azobenzene-derivative (AzD) bound by double-strand DNA, that by single-strand DNA and Azd shows trans-to-cis photoisomerization rate per pulse in the former is much lower than in the latter. © OSA / UP 2010.

DOI： 10.1364/up.2010.me6

Scopus

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

Scopus

PubMed

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

Scopus

PubMed

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

Scopus

PubMed

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

Scopus

PubMed

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

Scopus

PubMed

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

Scopus

Language：Japanese  

CiNii Research

Language：Japanese   Publisher：Chemical Communications  

The introduction of methyl groups into two ortho positions (2′ and 6′ positions) of the same benzene ring in an azobenzene remarkably raised both its photoregulation ability and the thermal stability of the cis-form. © The Royal Society of Chemistry.

DOI： 10.1039/b708952j

Scopus

PubMed

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

Scopus

PubMed

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.

Scopus

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.

Scopus

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.

Scopus

Language：Japanese   Publisher：Polymer Preprints, Japan  

We have introduced various dyes into DNA through D-threoninol and successfully functionalized DNA. However, little has been known on the relationship between the structure of intercalators and the stability of duplex. In this study, various intercalators (1 to 7 as shown in Scheme 1) were incorporated into DNA and the melting temperatures (TmS) of duplexes were measured. As shown in Table 1, azobenzene (1) stabilized duplex strongly compared with native duplex. However, larger molecules (2 and 3) and a smaller molecule (4) did not stabilize duplex. Therefore, molecular size is one of the most significant factors for the duplex stability.

Scopus

Language：Japanese   Publisher：Polymer Preprints, Japan  

We prepared molecularly imprinted polymers of β-cyclodextrin using three bioactive oligopeptides as templates; (a) angiotensin I (10-mer), (b) γ-endorphin (17-mer), and (c) leucine-enkephalin (Leu-Enk: 6-mer). With both angiotensin I and Leu-Enk as the templates, highly precise imprinting effects were observed. The present finding strongly indicates that we can prepare artificial antibodies by imprinting of CyD to a conformationally restrained part of a protein.

Scopus

Language：Japanese   Publisher：Polymer Preprints, Japan  

In the previous study, we introduced cationic dyes (Naphthyl Reds) and spacers alternately into the middle of 12mer oligonucleotides and prepared dye cluster by using DNA hybridization. The melting temperature (Tm) of this duplex was much higher than native DNA due to electrostatic and stacking interaction. Therefore, there is a possibility of forming dye cluster without the aid of natural bases. In this study, we decreased the number of natural bases at the terminal from 12 to 6 and 2 (see Scheme 1) and investigated the formation of dye cluster. Circular dichroism of 3C/3D and 3G/3G showed very strong symmetrical positive and negative Cotton effect at around 500 nm, suggesting the formation of dye cluster. The melting temperature (Tm) determined from the change of CD at 536 nm of 3C/3D was about 30°C, which almost coincided with the Tm from absorbance change at 260nm. Similarly, melting curve of 3C/3D of CD intensity at 536 nm also showed loose sigmoid, suggesting that 3G/3G also forms dye cluster.

Scopus

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.

Scopus

Language：Japanese   Publisher：Macromolecules  

Two kinds of vinyl monomers of β-cyclodextrin (β-CyD) that tether a vinyl group on either wider rim of the truncated cone or its smaller rim were synthesized and applied to the imprinting toward amino acid derivatives and oligopeptides in water. Mono-3-(N-acrylamido)-β-deoxy-altro-β- cyclodextrin (3-AAm-CyD) showed a remarkable imprinting effect for the enantioselective recognition of protected amino acids such as N-benzyloxycarbonyltyrosine (Z-Tyr). However, the imprinted polymer from mono-6-(N-acrylamido)-6-deoxy-βcyclodextrin (6-AAm-CyD) hardly showed enantioselectivity. According to NOESY analysis on the preorganized β-CyD/Z-Tyr complex in D2O, the aromatic moieties of Z-Tyr were included into the cavity of β-CyD from its wider rim. Since the vinyl group of 3-AAm-CyD protruded toward the template and was polymerized there, the detailed shape of the template was precisely copied on the polymer by the imprinting. In the case of 6-AAm-CyD, however, the shape of template could not be well transcribed because its vinyl group was located at opposite side of the cavity, and thus copolymerization occurred far from the template molecule. On the other hand, the imprinted polymers from both β-CyD vinyl monomers were effective for the recognition of sequences of tetrapeptides composed of two glycines and two phenylalanines, although the selectivity itself was not remarkable. In these polymers, even the β-CyD residues of 6-AAm-CyD were immobilized complementarily to the phenyl rings and bound them. © 2006 American Chemical Society.

DOI： 10.1021/ma060064f

Scopus

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

We have been investigating DNA state machines, especially those based on the opening of hairpin molecules in which state transitions are realized as hairpin loops are opened by molecules called openers. This paper introduces photo- and thermoregulation of such hairpin-based DNA machines, in which the openers become active by sensing external signals in the form of light or heat. We conducted fluorescence experiments and show that photo- and thermoregulation is possible. In the experiments, the openers become active when they are irradiated by UV light or when they receive heat as external input. For photoregulation, we use azobenzene-bearing oligonucleotides developed by the third author. © Springer-Verlag Berlin Heidelberg 2006.

DOI： 10.1007/11753681_26

Scopus

Language：Japanese   Publisher：Polymer Preprints, Japan  

We have successfully introduced functional molecules into DNA and prepared various aggregates by intercalation between base-pairs. Here, we synthesized DNA-pyrene conjugates through D-threoninol and investigated their properties. Excimer emission of pyrene was not observed when two pyrene moieties were intervened by one base-pair. On the other hand, strong excimer emission was observed at around 480 nm with complementary strand which lacks one base. Sequence dependence and linker modification are also investigated.

Scopus

CiNii Research

CiNii Research

Language：English  

DOI： 10.1021/ja037248j

PubMed

Language：English  

DOI： 10.1039/b302875e

PubMed

Language：English  

DOI： 10.1021/ja021153k

PubMed

Language：English  

DOI： 10.1093/nass/3.1.143

PubMed

Language：English  

DOI： 10.1093/nass/3.1.117

PubMed

Language：English  

DOI： 10.1093/nass/3.1.265

PubMed

Language：English  

DOI： 10.1002/1439-7633(20020802)3:8<786::AID-CBIC786>3.0.CO;2-P

PubMed

Language：English  

DOI： 10.1021/ja011988f

PubMed

Language：English  

DOI： 10.1021/ja011305w

PubMed

Language：English  

DOI： 10.1093/nass/2.1.75

PubMed

Language：English  

PubMed

Language：English  

DOI： 10.1002/1521-3773(20010716)40:14<2671::AID-ANIE2671>3.0.CO;2-Z

PubMed

Language：Japanese   Publisher：Journal of Immunology  

The protective roles of influenza viral nucleoprotein (NP), together with the cellular mechanism of the protection in the nasal site, were examined in BALB/c mice immunized intranasally with an adjuvant (cholera toxin B subunit containing 0.2% of the whole toxin)-combined A or B virus recombinant NP. The NP-immune mice, when challenged intranasally with a sublethal dose of the virus 3 wk after immunization, had accelerated virus clearance from the nasal site in both an influenza type-specific and a nonspecific manner, as shown by the protection from high morbidity from the second day after challenge. Both type-specific and nonspecific acceleration of recovery was confirmed by the increased survival rate after challenge with a lethal dose of virus in mice immunized and boosted with adjuvant-combined NP. The acceleration of nasal virus clearance was accompanied with acceleration of type-specific systemic delayed-type hypersensitivity (DTH) and with IFN-γ production by nasal lymphocytes. The nasal lymphocytes from the immunized and challenged mice generated a significantly high level of DTH when transferred locally, but no class I MHC-restricted CTL response. Moreover, nasal CD4+ T cells, induced by NP immunization and increased in number by the subsequent challenge, were involved in the accelerated IFN-γ production. These results suggest that nasal Th1 cells, capable of producing IFN-γ and mediating DTH, are involved in the type-specific acceleration of recovery from influenza after challenge in mice immunized intranasally with adjuvant-combined NP, although the nonspecific mechanism of accelerated recovery remains to be solved.

Scopus

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

DOI： doi: 10.1063/5.0192366

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

DOI： https://doi.org/10.1021/acssynbio.3c00459

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: doi.org/10.1039/D3CC05555H

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

DOI： DOI: doi.org/10.1246/bcsj.20230188

Authorship：Last author,　Corresponding author   Language：English  

DOI： DOI: doi.org/10.1021/acschembio.3c00353

Authorship：Last author   Language：English  

DOI： DOI: doi.org/10.1038/s41428-023-00776-7

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi.org/10.1002/chem.202300182

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: doi.org/10.1002/chem.202300182

Authorship：Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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

DOI： DOI: https://doi.org/10.1007/s00354-022-00173-3

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

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

DOI： DOI: https://doi.org/10.1038/s41428-022-00693-1

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI: 10.1039/d2ob00266c

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

DOI： doi.org/10.3390/mi13020193

Authorship：Last author,　Corresponding author   Language：English  

DOI： 10.1246/cl.210813

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

DOI： DOI:10.1246/cl.210788

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： DOI:10.1246/cl.210813

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

DOI： doi.org/10.3390/mi13020193

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

DOI： 10.1246/cl.210788

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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

Authorship：Last author,　Corresponding author   Language：English  

DOI： doi.org/10.3390/s21051785

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

DOI： 10.1002/chem.202102333

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

DOI： 10.1088/1361-6528/abef2c

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

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-1-4939-9216-4_17

Web of Science

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.201800020

Web of Science

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

DOI： 10.1002/cmdc.201700512

Web of Science

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.201700272

Web of Science

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

DOI： 10.1002/cbic.201700202

Web of Science

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

DOI： 10.1002/slct.201701126

Web of Science

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

DOI： DOI: 10.1039/c7ra06091b

Authorship：Lead author   Language：Japanese  

Authorship：Lead author   Language：Japanese  

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

DOI： 10.1093/nar/gkx200

Web of Science

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DOI： doi:10.1038/pj.2016.120

Authorship：Lead author   Language：Japanese  

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DOI： 10.1002/cbic.201600558

Web of Science

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Society of Polymer Science, Japan  

Natural nucleobases of DNA exhibit high duplex stability and high orthogonality, which are attractive from chemical viewpoints. If artificial base pairs are developed, it would be possible to develop novel functional materials. We have developed pseudo base pairs by incorporating non-natural molecules into DNA through <small>D</small>-threoninol. Although our pseudo bases are much different from natural nucleobases, they show high duplex stability and high orthogonality similarly to the natural ones. In this paper, we summarize our recent work on pseudo base pairs, focusing on the stability of DNA duplexes containing pseudo base pairs.

DOI： 10.1295/koron.2017-0009

Web of Science

Scopus

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DOI： 0000-0000

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DOI： DOI:10.1016/j.bmc.2016.06.055

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DOI： DOI: 10.1002/chem.201503303

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DOI： DOI: 10.1002/chem.201502653

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DOI： DOI: 10.1002/cbic.201500167

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DOI： DOI:10.1002/tcr.201402040

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DOI： DOI:10.1039/C3SC51197A

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DOI： 10.1002/chem.201301578

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DOI： 10.1016/j.bmc.2013.04.032

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DOI： 10.1016/j.optcom.2011.10.032

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An interstrand-wedged duplex involving alternating base pairs and covalently attached intercalators via D-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.

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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.

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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.

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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 &#61548;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.

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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.

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Using photoresponsive T7 promoters tethering two 2&#61602;-methylazobenzenes or 2&#61602;,6&#61602;-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&#61602;,6&#61602;-dimethylazobenzenes was 10-fold faster than that after visible light irradiation (400-600 nm). By attaching a non-modified azobenzene (Azo) and 2&#61602;,6&#61602;-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.

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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.

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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 (&#61508;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&#61602;,6&#61602;-dimethylazobenzene (2&#61602;,6&#61602;-Me-Azo), in which two ortho-positions of the distal benzene ring with respect to carboxyl group were methy-lated, exhibited the largest &#61508;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 &#61508;Tm.

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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.

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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 (&#61510;>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short F&ouml;rster radius (R0) of the donor pyrene.

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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 (&#61508;&#61548;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 &#61548;max at 336 nm and 480 nm in the single-strand, respectively, (&#61508;&#61548;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 &#61508;&#61548;max (82 nm) (&#61548;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 &#61508;&#61548;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

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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.

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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.

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“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.

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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.

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。

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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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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.

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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.

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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.

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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.

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.

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In this study, cationic dyes (methylstilbazole) were introduced into ODN. When two complementary ODNs, both of which tethered thise dye, were hybridized, the melting temperature drastically increased. Furthermore, The duplex was further stabilized by introducing multiple dyes.

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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.

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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.

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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.

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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.

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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.

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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&cent;-…pNpXpN…-3&cent; (p = phosphate; N = nucleoside; X = azobenzene residue) could be digested to pX (the phosphate at the 5&cent; 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.

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.

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.

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&cent;&reg;5&cent; 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.

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.

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.

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.

Authorship：Lead author   Language：English  

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.

Authorship：Lead author   Language：English  

Novel dye aggregates (“comb-type" aggregates) was prepared by hybridizing modified oligodeoxyribo-nucleotides (ODNs), in which dyes were introduced consecutively. From NMR structural analysis, dye molecules were intercalated between base pairs and stacked in anti-parallel manner. When ODNs containing three Methyl Red moieties were hybridized, strong exciton coupling was observed. In addition, thermal stability of duplex was substantially enhanced due to the intermolecular stacking.

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.

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.

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

The introduction of methyl groups into two ortho positions (2&cent; and 6&cent; positions) of the same benzene ring in an azobenzene remarkably raised both its photoregulation ability and the thermal stability of the cis-form.

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

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

For the detection of deletion polymorphisms, two pyrene moieties are tethered to oligodeoxyribonucleotide (ODN) on both sides of the intervening base. When this probe ODN was hybridized with wild type ODN, both pyrenes intercalated between base pairs and only monomer emission was observed. In contrast, strong excimer emission was generated by hybridization with one-base deletion mutant. Similarly, two-base deletion was also detected.

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

Ordered dye cluster of Methyl Reds was formed in double-stranded DNA by hybridizing two complementary DNA-dye conjugates that involve Methyl Red moiety on threoninol linker and 1,3-propanediol as a spacer alternately in the middle of the sequence. In the duplex, Methyl Reds from each strand were axially stacked anti-parallel to each other as determined from NMR analysis. This clustering of Methyl Reds induced distinct change of both UV-Vis and CD spectra. Single-stranded DNA-Methyl Red conjugate on D-threoninol linkers and (1,3-propanediol) spacers exhibited broad absorption spectrum having lmax at around 480 nm, and almost no CD was observed at around absorption maximum of Methyl Red. However, when Methyl Reds were clustered by hybridization, lmax shifted towards shorter wavelength with respect to its monomeric transition. This hypsochromic shift increased with the number of Methyl Reds. Furthermore, positive couplet was also strongly induced here. These dye clusters are H-aggregates, in which molecular excitons are coupled. The positive couplet demonstrates that the clusters on D-threoninol forms right-hand helix. In contrast, induced CD became much weaker with Methyl Red on L-threoninol, which intrinsically prefers counterclockwise winding. Thus, mutual orientation of the stacked dyes was controlled by the chirality of the linker.

Authorship：Lead author   Language：English  

Authorship：Lead author   Language：English  

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

By introducing an intercalator through D-threoninol to the 10-23 DNAzyme at the junction between its catalytic loop and the binding arm, the RNA cleavage activity was greatly improved.

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

Azobenzene was additionally introduced into side chain of T7 promoter for the photoregulation of transcription reaction by T7 RNA polymerase (T7 RNAP). When single azobenzene molecule was introduced into the T7 promoter either at the loop binding region of the RNAP (–7 to –11 position) or at the unwinding region (+1 to –4 position), transcription was suppressed in the trans-form but proceeded faster in the cis-form. The amount of transcripts after UV irradiation with respect to that under dark was only 1.5 – 2.0-fold. Kinetic analysis of the transcription reaction revealed that photoregulatory mechanism was different in these positions. The photoisomerization of an azobenzene at the loop binding region primarily affected Km. On the other hand, the isomerization of an azobenzene at unwinding region mainly affected kcat. Still more clear-cut photoregulation was achieved when two azobenzenes were introduced into both loop binding and unwinding regions, respectively: transcription proceeded 7.6-fold faster after UV irradiation than that under dark. This synergistic effect was observed only when two azobenzenes were introduced into these two different regions, respectively, and introduction into the same loop binding region drastically lowered the transcription activity. The cooperation of two azobenzenes at loop binding and unwinding region would contribute to the clear-cut photoregulation of transcription.

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

Two kinds of vinyl monomers of b-cyclodextrin (b-CyD) that tether a vinyl group on either wider rim of the truncated cone or its smaller rim were synthesized and applied to the imprinting towards amino acid derivatives and oligopeptides in water. Mono-3-(N-acrylamido)-3-deoxy-altro-b-cyclodextrin (3-AAm-CyD) showed remarkable imprinting effect for the enantioselective recognition of protected amino acids such as N-benzyloxycarbonyltyrosine (Z-Tyr). However, the imprinted polymer from mono-6-(N-acrylamido)-6-deoxy-b-cyclodextrin (6-AAm-CyD) hardly showed enantioselectivity. According to NOESY analysis on pre-organized b-CyD/Z-Tyr complex in D2O, the aromatic moieties of Z-Tyr were included into the cavity of b-CyD from its wider rim. Since the vinyl group of 3-AAm-CyD protruded towards the template and was polymerized there, detailed shape of the template was precisely copied on the polymer by the imprinting. In case of 6-AAm-CyD, however, the shape of template could not be well transcribed because its vinyl group was located at opposite side of the cavity and thus co-polymerization occurred far from the template molecule. On the other hand, the imprinted polymers from both b-CyD vinyl monomers were effective for the recognition of sequences of tetrapeptides composed of two glycines and two phenylalanines, although the selectivity itself was not remarkable. In these polymers, even the b-CyD residues of 6-AAm-CyD were immobilized complementarily to the phenyl rings and bound them.

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

By using N-(3-triethoxysilyl)propylacrylamide (TPAAm), vinyl groups were introduced onto the surface of silicagel.
On the surface of this silica-gel, b-CyD was molecularly imprinted by using a redox initiator, and the composite
was used as stationary phase of high performance liquid chromatography (HPLC). The pump pressure was sufficiently
low and did not increase even after continuous elution for 24 h. In order to prepare still more stable columns,
a new polymerization process was developed. There, the redox initiator was first mixed with the surface-modified
silica-gel and then vinylated b-CyD, crosslinker, and the template were added. This modification promoted the
immobilization of b-CyD copolymer to the silica-gel, resulting in still lower pump pressure. Concurrently, the
imprinting efficiency was increased in comparison with previous method where the redox initiator was directly
added to the mixture of the b-CyD–template complex, crosslinker, and surface-modified silica-gel. The molecularly
imprinted b-CyD column, prepared by this new method, efficiently discriminated the enantiomers of
N-benzyloxycarbonyltyrosine.

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

Exciplex emission from pyrene and N,N-dimethylaniline moieties was observed in aqueous solution by incorporating them into DNA. By using exciplex formation, one-base deletion was successfully detected.

Authorship：Lead author   Language：English  

Authorship：Lead author   Language：English  

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

New hetero aggregates in which Methyl Reds and Naphthyl Reds are stacked alternately were successfully prepared by hybridization of two DNA-dye conjugates. When the Naphthyl Red conjugate was hybridized with Methyl Red conjugate, a new sharp absorption band (H-band) appeared at 480 nm that was different from the lmax of each dye conjugate in the single-stranded state. In addition to this new band in the UV-Vis spectrum, strong circular dichroism was also induced by interstrand hetero-stacking of these dyes. These results demonstrated that even different dyes as well as identical dyes could exhibit H-band.

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

Interstrand H-aggregates of cationic dyes are prepared by hybridization of the two DNA-dye conjugates involving Naphthyl Red moiety and spacer alternately in the middle of the sequence. At pH 5.0 where Naphthyl Red is positively charged, single-stranded DNA-Naphthyl Red conjugate involving three dye groups and three spacer residues exhibited broad absorption spectrum having lmax at around 530 nm. But hybridization of two DNA-Naphthyl Red conjugates that are complementary provided rather different spectrum: much narrower absorption band appeared at 507 nm, which is 23 nm shorter than the single stranded conjugates. These spectroscopic behaviors indicate that dyes in the duplex are H-aggregated and excitons are strongly coupled in the aggregate. In addition to the appearance of narrow H-band, melting temperature dramatically increased by the H-aggregation of stacked cationic dyes compared with that of natural duplex without dye and spacer residues. Thus, positive charges on the stacked dyes did not interfere the duplex formation by the electrostatic repulsion, and moreover fairly promoted the hybridization.

Authorship：Lead author   Language：English  

Authorship：Lead author   Language：English  

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

RNA digestion by RNase H, which is responsible for the antisense effect, was efficiently photo-regulated by use of the duplex of azobenzene-tethered sense DNA and native antisense DNA. Under dark, RNA digestion was suppressed because antisense DNA was strongly hybridized with azobenzene-tethered sense DNA, and accordingly RNA was isolated. On UV light irradiation, antisense DNA was released from the azobenzene-tethered DNA due to the trans to cis isomerization and hybridized with RNA, which was digested by RNase H.

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

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これまでに様々な蛍光色素で修飾した核酸プローブが合成されており、溶液中での遺伝子多型の検出に関しては数多くの報告がなされてきた。しかしながら、それに比べて基板にこれらの核酸プローブを固定化した例は必ずしも多くない。その原因としては、プローブ自身の蛍光によるバックグラウンドが挙げられる。蛍光色素で修飾した核酸プローブを基板上に固定化する場合、プローブ自身が発する蛍光により検出が阻害されてしまう。そのため、蛍光性核酸プローブを固定化したDNAチップを調製するためには、ターゲット非存在下でプローブ自身の蛍光を抑制することが必要となる。そこで、本節ではこのようなプローブ単体でのバックグラウンドが抑制された核酸プローブの例として１）モレキュラービーコン及び２）レシオメトリックプローブについて述べる。

Responsible for pages：603-610   Language：Japanese

これまでに様々な蛍光色素で修飾した核酸プローブが合成されており、溶液中での遺伝子多型の検出に関しては数多くの報告がなされてきた。しかしながら、それに比べて基板にこれらの核酸プローブを固定化した例は必ずしも多くない。その原因としては、プローブ自身の蛍光によるバックグラウンドが挙げられる。蛍光色素で修飾した核酸プローブを基板上に固定化する場合、プローブ自身が発する蛍光により検出が阻害されてしまう。そのため、蛍光性核酸プローブを固定化したDNAチップを調製するためには、ターゲット非存在下でプローブ自身の蛍光を抑制することが必要となる。そこで、本節ではこのようなプローブ単体でのバックグラウンドが抑制された核酸プローブの例として１）モレキュラービーコン及び２）レシオメトリックプローブについて述べる。

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天然のDNAの更なる機能化を目的としてこれまでに様々な化学修飾がDNAに対して行われてきたが、上述のようにDNAの応用分野が多様化したことで、DNAの化学修飾の重要性は更に増しつつある。もちろん天然の４つの核酸塩基だけでも多彩なナノ構造を形成し様々な機能を発揮するが、化学修飾によって天然のDNAでは実現不可能な機能を補うことが出来れば、DNAの応用範囲は飛躍的に拡大するであろう。本稿ではこのような観点から筆者らが開発したアゾベンゼン導入型光応答性DNAと、これを用いたDNAナノマシンのダイナミックな光制御について解説したい。

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Responsible for pages：47-64, 119-138   Language：English

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Responsible for pages：147   Language：English

Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

For a signal amplification system that is orthogonal to DNA, we designed a simplified seesaw gate composed of only D-aTNA. This new system performed signal amplification by toehold exchange reaction just as the DNA circuit did. Moreover, the D-aTNA circuit was not affected by natural nucleic acids carrying sequences complementary to the D-aTNA. In the presence of an SNA interface, however, an RNA signal was converted to D-aTNA signal, resulting in successful activation of D-aTNA circuit. This system can be used to design signal-amplification circuits that are not influenced by contaminating DNA and RNA.

DOI： 10.1002/slct.201701126

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：OXFORD UNIV PRESS  

Differences in structures and flexibilities of DNA duplexes play important roles on recognition by DNA-binding proteins. We herein describe a novel method for structural analyses of DNA duplexes by using orientation dependence of Forster resonance energy transfer (FRET). We first analyzed canonical B-form duplex and correct structural parameters were obtained. The experimental FRET efficiencies were in excellent agreement with values theoretically calculated by using determined parameters. We then investigated DNA duplexes with nick and gaps, which are key intermediates in DNA repair systems. Effects of gap size on structures and flexibilities were successfully revealed. Since our method is facile and sensitive, it could be widely used to analyze DNA structures containing damages and non-natural molecules.

DOI： 10.1093/nar/gkx200

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Nature Publishing Group  

Further development of DNA nanotechnology requires new functional oligonucleotides composed of nucleobases beyond the native four. In this review, we demonstrate new methodology for DNA and RNA functionalization using a base surrogate prepared from d-threoninol (2-amino-1,3-butanediol). Using this nucleobase surrogate, we can introduce functional molecules at any position of the sequence. Our methodology is conceptually similar to the copolymerization of multiple monomers: phosphoramidite monomers corresponding to the base surrogate and natural nucleotides are copolymerized on a solid support to prepare the functional oligonucleotides. Copolymerization allows for stable functional motifs, including wedges, interstrand-wedges, dimers and clusters. By selecting suitable functional molecules and motifs, we can design photofunctional oligonucleotides, such as: (1) photoresponsive DNA that enables reversible formation and dissociation of the duplex by photoirradiation
(2) [2+2] photocycloaddition of stilbene derivatives
(3) orientation-dependent FRET (fluorescence (Förster) resonance energy transfer) systems
(4) sequence-specific fluorescent probe for the detection of DNA and RNA
and (5) functional siRNA for fluorescent labeling of mature RISC (RNA-induced silencing complex).

DOI： 10.1038/pj.2016.120

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Acyclic serinol derivatives are useful scaffolds for tethering dyes within DNA duplexes. Here we synthesised an inverse L-threoninol (il-threoninol) scaffold and compared its effect on DNA duplex stability to other acyclic artificial nucleic acid scaffolds that are based on D-threoninol, L-threoninol, and serinol. When planar trans-azobenzene was incorporated into the DNA duplex through a single bulge-like motif (the wedge), the il-threoninol scaffold stabilised the duplex most efficiently. When scaffolds were incorporated in complementary positions (dimer motif) or in three adjacent positions (cluster motif), D-threoninol was the most stabilising. CD spectra indicated that the effect of scaffold on the duplex stability was closely related to the winding induced by each scaffold. When trans-azobenzene was photo-isomerised to non-planar cis-azobenzene, il-threoninol destabilised the duplex most strongly, irrespective of the number of artificial residues incorporated. The properties of the il-threoninol scaffold make it a useful tether for dyes or other functionalities.

DOI： 10.1002/cbic.201600558

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Serinol nucleic acid (SNA) is a novel nucleic acid analogue that can form highly stable heteroduplexes with complementary DNA and RNA sequences. Structurally, SNA is a close mimic to peptide nucleic acid (PNA) which is widely used in diagnostic and therapeutic applications. SNA chemistry is relatively new, and so far the scope of SNA has only been explored in improving the efficacy of small interfering RNA and for developing a highly sensitive molecular beacon for diagnostic applications. In this study, we investigated the potential of SNA-modified antisense oligonucleotide (AO) in parallel to PNA-oligo for splicemodulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20mer SNA and PNA antisense oligonucleotide (AO) designed to induce exon-23 skipping in the mouse dystrophin gene transcript. Our results demonstrated that the SNA AO induced exon-23 skipping at all tested concentrations, whereas the corresponding PNA AO failed to induce any exon-23 skipping upon 24 hours of transfection using Lipofectin transfection reagent. Our results further expands the potential of SNA oligonucleotides in therapeutic applications.

DOI： 10.1039/c7ra06091b

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Serinol nucleic acid (SNA) is a novel nucleic acid analogue that can form highly stable heteroduplexes with complementary DNA and RNA sequences. Structurally, SNA is a close mimic to peptide nucleic acid (PNA) which is widely used in diagnostic and therapeutic applications. SNA chemistry is relatively new, and so far the scope of SNA has only been explored in improving the efficacy of small interfering RNA and for developing a highly sensitive molecular beacon for diagnostic applications. In this study, we investigated the potential of SNA-modified antisense oligonucleotide (AO) in parallel to PNA-oligo for splicemodulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20mer SNA and PNA antisense oligonucleotide (AO) designed to induce exon-23 skipping in the mouse dystrophin gene transcript. Our results demonstrated that the SNA AO induced exon-23 skipping at all tested concentrations, whereas the corresponding PNA AO failed to induce any exon-23 skipping upon 24 hours of transfection using Lipofectin transfection reagent. Our results further expands the potential of SNA oligonucleotides in therapeutic applications.

DOI： 10.1039/c7ra06091b

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

We report on the characterization of a novel hetero-selective DNA-like duplex of pyrene and anthraquinone pseudo base pairs. The pyrene/anthraquinone pairs showed excellent selectivity in hetero-recognition and even trimers were found to form a hetero-duplex. Pyrene and anthraquinone moieties were tethered on acyclic D-threoninol linkers and linked to adjacent residues by using standard phosphoramidite chemistry. When pyrene and anthraquinone were incorporated at pairing positions in complementary strands of natural DNA oligonucleotides, the duplex was stabilized significantly. Moreover, a pyrene hexamer and an anthraquinone hexamer formed a stable artificial hetero-duplex without the assistance of natural base pairs. The pyrene/anthraquinone pair was so stable that even trimers formed a hetero-duplex under conditions in which natural DNA strands of three residues do not.

DOI： 10.1002/chem.201502653

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

An artificial nucleic acid based on acyclic serinol building blocks and termed serinol nucleic acid (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA-MB could detect its complementary RNA with extremely high sensitivity; the signal-to-background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA-MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA-MB has potential for use as a biological tool capable of visualizing RNA in living cells.

DOI： 10.1002/cbic.201500167

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Here, we investigated spectroscopic behaviors of tetramethylrhodamine (TMR) homo- and hetero-dimers within DNA duplex. In order to shield the chromophores from natural base pairs, we used cyclohexyl base pairs as 'insulators'; these pairs were inserted between the chromophores and nucleobases. When a single TMR moiety was sandwiched between cyclohexyl base pairs, the emission intensity increased by fivefold relative to a TMR between natural base pairs, because electron transfer from nucleobases was suppressed. Next, we inserted two TMRs between the cyclohexyl base pairs and found that they facilitated H-dimer formation of TMR; a distinct hypsochromic shift was induced only when cyclohexyl base pairs were inserted. We further examined quenching behavior of a TMR paired with a quencher dye between cyclohexyl base pairs. Interestingly, fluorescence from TMR was quenched by nitro methyl red more efficiently in the presence of cyclohexyl base pairs than in their absence. This suggests that neighboring natural base pairs disturbed electron or hole transfer between the fluorophore and the quencher. The cyclohexyl base pairs shielded the chromophore pair from the natural base pairs and allowed intrinsic electron transfer. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2013.04.032

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

DOI： 10.1002/chem.201301578

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

RNA interference (RNAi) is an endogenous gene silencing system that has been harnessed to inhibit expression of specific genes through the introduction of short double-stranded RNAs, called small interfering RNAs (siRNAs) into cells. After entry into a cell, an siRNA is assembled into the RNA-induced silencing complex (RISC) and suppresses the target gene translation through interaction between the antisense (guide) strand of the siRNA and the target mRNA. To evaluate the intracellular fate of siRNAs, we performed imaging analyses using siRNAs labeled with newly designed fluorophore-quencher clusters introduced through the base surrogate with D-threoninol as a scaffold. Fluorescence microscopy analyses indicated that mature RISC containing fluorescently labeled antisense strand was localized within P-bodies. Thus, the antisense strand uptake into the RISC machinery was successfully visualized.

DOI： 10.1039/c3sc51197a

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The femtosecond photoisomerization processes of trans (T) 4-carboxy-2',6'-dimethylazobenzen, which has been employed recently as an efficient photoregulator of DNA hybridization, were clarified by the rate equation analysis of measured transient absorbance changes with (350 nm) and without (380 nm) ground-state absorption of both the reactant (T) and photoproduct (cis: C) isomers under S-2(T)-band excitation (360 nm, 150 fs pump): after excitation to the S-2(T) state with a 450-fs lifetime, similar to 1.5% of the 1-molecules in the S-2(T) state are isomerized to the c-form within similar to 6 ps through the intermediate state (so called bottleneck state), but most of those return back to the T ground-state S-2(T) via the internal conversion processes with an ultrafast kinetic rate of 2.2 x 10(12) s(-1). Moreover, the rate equation analysis enables us to determine the T-to-C photoisomerization rate eta(T,C) per pump pulse to be 0.0011 at the pump energy of 80 nJ from the amplitude A(3,350) of the offset component in the 350-nm probe signal, and to obtain the photoisomerization quantum yield Phi(T,C) = 0.094. The latter value is slightly lower than that of T-azobenzene, and well agrees with that (Phi(T,C) = 0.097) measured by the conventional CW irradiation method using a photostationary state. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.optcom.2011.10.032

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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

Language：English   Publishing type：Research paper, summary (national, other academic conference)  

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&amp;cent;&amp;reg;5&amp;cent; 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.

Event date： 2019.12

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Guangzhou   Country：China  

Event date： 2019.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2019.10

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Koganei Civic Center   Country：Japan  

Event date： 2019.8

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Taiwan, Province of China  

Event date： 2019.7

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Hotel Hokke Club Hakodate   Country：Japan  

Event date： 2019.6

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Shenyang Pharmaceutical University   Country：China  

Event date： 2019.6

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2019.6

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Brussels, Belgium   Country：Belgium  

Event date： 2018.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Australia  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2018.9

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Hotel Grand Hyatt, Goa, India   Country：India  

Event date： 2018.7

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：仙台   Country：Japan  

Event date： 2018.5

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：グランフロント大阪 ナレッジキャピタル   Country：Japan  

Event date： 2018.5

Language：English   Presentation type：Oral presentation (keynote)  

Venue： National Taiwan University of Science and Technology, Taipei (Tiwan)   Country：Taiwan, Province of China  

Event date： 2018.3

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2018.3

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Siem Reap, Cambodia   Country：Cambodia  

Event date： 2018.2

Language：English   Presentation type：Oral presentation (invited, special)  

Venue： Hat Yai, Songkhla (Thailand)   Country：Thailand  

Event date： 2017.12

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：IIT Bombay (India)   Country：India  

Event date： 2017.12

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Kyoto Univ. (Kyoto)   Country：Japan  

Event date： 2017.10

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Tokyo institute of Technology   Country：Japan  

Event date： 2017.7

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2017.7

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Takayama, Gifu   Country：Japan  

Event date： 2016.12

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Osaka, Japan   Country：Japan  

Event date： 2016.10

Language：English   Presentation type：Oral presentation (invited, special)  

Venue： Prien (Chiemsee), Germany   Country：Germany  

Event date： 2016.9

Language：English   Presentation type：Oral presentation (invited, special)  

Venue： Fukuoka, Japan   Country：Japan  

Event date： 2016.7

Language：English   Presentation type：Oral presentation (invited, special)  

Country：China  

Event date： 2016.7

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2016.5

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：神戸国際会議場・展示場　神戸   Country：Japan  

Event date： 2015.12

Language：English   Presentation type：Oral presentation (invited, special)  

Country：United States  

Event date： 2015.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：China  

Event date： 2015.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Thailand  

Event date： 2015.6

Language：English   Presentation type：Oral presentation (invited, special)  

Country：China  

Event date： 2014.9

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Portugal  

Event date： 2014.7

Language：English   Presentation type：Oral presentation (invited, special)  

Country：United Kingdom  

Event date： 2014.6

Language：English   Presentation type：Oral presentation (general)  

Country：Czech Republic  

Event date： 2014.5

Language：English   Presentation type：Oral presentation (invited, special)  

Country：China  

Event date： 2013.12

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Germany  

Event date： 2013.12

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Germany  

Event date： 2013.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Korea, Republic of  

Event date： 2013.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2013.8 - 2013.9

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2013.2

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2012.12

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2012.7 - 2012.8

Language：English   Presentation type：Oral presentation (invited, special)  

Country：United States  

Event date： 2012.6

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Italy  

Event date： 2012.5

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Korea, Republic of  

Event date： 2012.1

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2011.11

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2011.10

Language：English   Presentation type：Oral presentation (invited, special)  

Country：China  

Event date： 2011.6

Language：English   Presentation type：Oral presentation (keynote)  

Country：Czech Republic  

Event date： 2011.1

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2010.11

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2010.10

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2010.10

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2010.8

Language：English   Presentation type：Oral presentation (invited, special)  

Event date： 2010.6

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2010.6

Language：English   Presentation type：Oral presentation (invited, special)  

Event date： 2009.12

Language：English   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2009.12

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2009.9

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Country：Japan  

Event date： 2009.9