研究者詳細 - 浅沼　浩之

Perylene-Cy3 FRET System to Analyze Photoactive DNA Structures 査読有り

Kawai H., Doi T., Ito Y., Kameyama T., Torimoto T., Kashida H., Asanuma H.

Chemistry - A European Journal 27 巻 ( 50 ) 頁： 12845 - 12850 2021年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers 招待有り査読有り

Murayama K., Asanuma H.

ChemBioChem 22 巻 ( 15 ) 頁： 2507 - 2515 2021年8月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Dual Crosslinking Photo-Switches for Orthogonal Photo-Control of Hybridization Between Serinol Nucleic Acid and RNA 査読有り

Yamano Y., Murayama K., Asanuma H.

Chemistry - A European Journal 27 巻 ( 14 ) 頁： 4599 - 4604 2021年3月

担当区分：最終著者,　責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemistry - A European Journal

Wavelength-selective photo-regulation by multiple chromophores responding to different wavelengths can expand the variation of photo-manipulating systems. Herein, we report the orthogonal photo-regulation of duplex formation between serinol nucleic acid (SNA) and RNA using light-induced crosslinking reactions mediated by a new photo-reactive nucleobase 8-naphthylvinyladenine (NVA) and previously described 8-pyrenylvinyladenine (PVA). An intrastrand crosslink was induced in an SNA strand containing two adjacent NVA residues by irradiation with 340–405 nm light; the crosslink was reversed by irradiation with ≤300 nm light. In an SNA strand with adjacent NVA and PVA residues, an intrastrand crosslink resulted from irradiation with 405–465 nm light that was reversed by irradiation with ≤340 nm light. Intrastrand photo-crosslinking caused severe destabilization of an SNA/RNA duplex, resulting in dissociation to single strands. Cycloreversion resulted in duplex formation. With these NVA/NVA and NVA/PVA photo-switches, four hybridization states of two SNA/RNA duplexes could be orthogonally photo-controlled by irradiation with a suitable wavelength of light.

DOI： 10.1002/chem.202003528

A helical amplification system composed of artificial nucleic acids 査読有り

Kashida H., Nishikawa K., Shi W., Miyagawa T., Yamashita H., Abe M., Asanuma H.

Chemical Science 12 巻 ( 5 ) 頁： 1656 - 1660 2021年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Science

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time. It is usually impossible to prepare achiral nanostructures composed of nucleic acids because of their intrinsic chirality. We used serinol nucleic acid (SNA) oligomers for the preparation of achiral nanowires because SNA oligomers with symmetrical sequences are achiral. Nanowire formation was confirmed by atomic force microscopy and size exclusion chromatography. When a chiral nucleic acid with a sequence complementary to SNA was added to the nanostructure, helicity was induced and a strong circular dichroism signal was observed. The SNA nanowire could amplify the helicity of chiral nucleic acids through nucleobase stacks. The SNA nanostructures have potential for use as platforms to detect chiral biomolecules under aqueous conditions because SNA can be readily functionalized and is water-soluble.

DOI： 10.1039/d0sc05245k

Nonenzymatic polymerase-like template-directed synthesis of acyclic L-threoninol nucleic acid 査読有り

Murayama, K.; Okita, H.; Kuriki, T.; Asanuma, H.

Nature Communications 12 巻頁： 804 2021年2月

Light-Regulated Liquid-Liquid Phase Separation for Spatiotemporal Protein Recruitment and Cell Aggregation 査読有り

Ikeuchi N., Komachi T., Murayama K., Asanuma H., Maruyama A., Shimada N.

ACS Applied Materials and Interfaces 13 巻 ( 4 ) 頁： 5652 - 5659 2021年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Applied Materials and Interfaces

We have previously shown that the upper critical solution temperature-type thermoresponsive ureido polymers such as polyallylurea and poly(2-ureidoethylmethacrylate) derivatives show liquid-liquid phase separation (LLPS), also known as simple coacervation, under physiological conditions below their phase-separation temperatures (Tp). The addition of the polymer-rich coacervate droplets that result from LLPS to a monolayer cell culture induced aggregation of cells into multicellular spheroids. In this study, we prepared a ureido copolymer, poly(vinylamine-co-vinylurea), with azobenzene substituents (Azo-PVU) and demonstrated light-guided assembly and disassembly of LLPS coacervates. Azo-PVUs with Tp values ranging from 10 to 52 °C were prepared by changing the azobenzene content. Ultraviolet light caused a decrease in the Tp of Azo-PVU because of trans-to-cis photoisomerization of the azobenzene and irradiation with visible light increased the Tp. Thus, LLPS of Azo-PVU was reversibly controlled. The coacervate droplets deposited on a dish surface were immediately dissolved by targeted UV irradiation (owing to a decrease in the Tp). Spatially controlled recruitment of proteins on the dish surface was achieved when protein solution was added to the light-patterned surface. Furthermore, the light-guided deposition of coacervates resulted in the spatiotemporal transformation of monolayer cells to aggregates. This light-controlled LLPS will allow the preparation of novel liquid-based materials for biomolecular and cellular engineering.

DOI： 10.1021/acsami.0c22314

Quantitative Analyses of Förster Resonance Energy Transfer between Identical Pyrene Chromophores (Homo-FRET) In DNA Scaffolds. 査読有り

Kashida H., Kawai H., Azuma H., Araki Y., Wada T., Asanuma H.

ChemPhotoChem 5 巻 ( 2 ) 頁： 167 - 172 2021年2月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemPhotoChem

Förster resonance energy transfer between identical chromophores (homo-FRET) has been difficult to analyze since neither emission intensity nor lifetime changes with the occurrence of homo-FRET. Herein we used a DNA scaffold to analyze homo-FRET between pyrene moieties. The DNA scaffold was modified with two pyrenes and a quencher, anthraquinone. Homo-FRET was detected by monitoring quenching of pyrene emission and the decrease in the fluorescence lifetime of pyrene. Homo-FRET efficiencies could be calculated by excluding effects of hetero-FRET. The experimentally determined efficiencies showed an excellent agreement with Förster theory. These results will inform design of novel molecular probes and light-harvesting antennae.

DOI： 10.1002/cptc.202000199

A Pyrene-Modified Serinol Nucleic Acid Nanostructure Converts the Chirality of Threoninol Nucleic Acids into Circularly Polarized Luminescence Signals 査読有り

Kashida H., Nishikawa K., Ito Y., Murayama K., Hayashi I., Kakuta T., Ogoshi T., Asanuma H.

Chemistry - A European Journal 2021年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

Intrastrand backbone-nucleobase interactions stabilize unwound right-handed helical structures of heteroduplexes of L-aTNA/RNA and SNA/RNA 査読有り

RNA以前に非環状型XNAがPre-RNAとして存在したか？招待有り査読有り

浅沼浩之, 沖田ひかり, 村山恵司

Viva Origino 49 巻 ( 3 ) 頁： 9 2021年

Kamiya Y., Satoh T., Kodama A., Suzuki T., Murayama K., Kashida H., Uchiyama S., Kato K., Asanuma H.

Communications Chemistry 3 巻 ( 1 ) 頁： 156 2020年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Communications Chemistry

Xeno nucleic acids, which are synthetic analogues of natural nucleic acids, have potential for use in nucleic acid drugs and as orthogonal genetic biopolymers and prebiotic precursors. Although few acyclic nucleic acids can stably bind to RNA and DNA, serinol nucleic acid (SNA) and L-threoninol nucleic acid (L-aTNA) stably bind to them. Here we disclose crystal structures of RNA hybridizing with SNA and with L-aTNA. The heteroduplexes show unwound right-handed helical structures. Unlike canonical A-type duplexes, the base pairs in the heteroduplexes align perpendicularly to the helical axes, and consequently helical pitches are large. The unwound helical structures originate from interactions between nucleobases and neighbouring backbones of L-aTNA and SNA through CH–O bonds. In addition, SNA and L-aTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA. The unique structural features of the RNA-recognizing mode of L-aTNA and SNA should prove useful in nanotechnology, biotechnology, and basic research into prebiotic chemistry.

DOI： 10.1038/s42004-020-00400-2

Investigation of strand-selective interaction of SNA-modified siRNA with AGO2-MID 査読有り

Kamiya Y., Takeyama Y., Mizuno T., Satoh F., Asanuma H.

International Journal of Molecular Sciences 21 巻 ( 15 ) 頁： 1 - 13 2020年8月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Molecular Sciences

Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5’-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5’ end of the passenger strand.

DOI： 10.3390/ijms21155218

Photo-regulated trajectories of gliding microtubules conjugated with DNA 査読有り

Akter M., Keya J.J., Kabir A.M.R., Asanuma H., Murayama K., Sada K., Kakugo A.

Chemical Communications 56 巻 ( 57 ) 頁： 7953 - 7956 2020年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

We regulate the persistency in motion of kinesin-driven microtubules (MTs) simply using a photoresponsive DNA (pDNA) and ultraviolet (UV)-visible light. The path persistence length of MTs, which is a measure of the persistency in their motion, increases and decreases upon illuminating the MTs with UV and visible light respectively. Moreover, pDNA is found to work as a shield for MTs against damage under UV irradiation.

DOI： 10.1039/d0cc03124k

Efficient Light-Harvesting Antennae Resulting from the Dense Organization of Dyes into DNA Junctions through d-Threoninol 査読有り

Kashida H., Azuma H., Maruyama R., Araki Y., Wada T., Asanuma H.

Angewandte Chemie - International Edition 59 巻 ( 28 ) 頁： 11360 - 11363 2020年7月

担当区分：最終著者,　責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Angewandte Chemie - International Edition

Herein we report the construction of efficient light-harvesting antennae by hybridization of DNA oligonucleotides containing high densities of fluorophores into DNA junctions through d-threoninol. Six pyrene donors could be incorporated into each arm without self-quenching. A perylene acceptor was located at the center of the junction. Antenna effects of a duplex and three- to eight-way junctions were systematically compared. Six- and eight-way junctions had the highest antenna effects, and their effective absorption coefficients were 8.5 times higher than that of perylene. Interestingly, even-numbered junctions had higher efficiencies than odd-numbered junctions. Nondenaturing gel analyses and fluorescence lifetime measurements demonstrated that the strong odd–even effects were derived from differences in the stability of junctions. The results presented will guide the design of efficient artificial photosynthetic systems.

DOI： 10.1002/anie.202004221

A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity 査読有り

Chen Y., Murayama K., Kashida H., Kamiya Y., Asanuma H.

Chemical Communications 56 巻 ( 40 ) 頁： 5358 - 5361 2020年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

A triplex-forming oligonucleotide (TFO) linear probe containing perylene derivatives was synthesized. The TFO linear probe formed a remarkably stable triplex with a target DNA duplex, resulting in the light-up of fluorescence emission. The sensitivity was extremely high even at pH 7. Detection of PCR-amplified target DNA was demonstrated.

DOI： 10.1039/d0cc01865a

Incorporation of Pseudo-complementary Bases 2,6-Diaminopurine and 2-Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization 査読有り

Kamiya Y., Sato F., Murayama K., Kodama A., Uchiyama S., Asanuma H.

Chemistry - An Asian Journal 15 巻 ( 8 ) 頁： 1266 - 1271 2020年4月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemistry - An Asian Journal

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.

DOI： 10.1002/asia.201901728

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue 査読有り

Murayama K., Asanuma H.

ChemBioChem 21 巻 ( 1-2 ) 頁： 120 - 128 2020年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemBioChem

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without PeU modification; thus suggesting that structural distortion is not induced by the modification. If two PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.

DOI： 10.1002/cbic.201900498

8-Pyrenylvinyl Adenine Controls Reversible Duplex Formation between Serinol Nucleic Acid and RNA by [2 + 2] Photocycloaddition 査読有り

Murayama K., Yamano Y., Asanuma H.

Journal of the American Chemical Society 141 巻 ( 24 ) 頁： 9485 - 9489 2019年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of the American Chemical Society

Photocontrol of duplex formation between the totally artificial serinol nucleic acid (SNA) and target RNA was made possible using a photoresponsive nucleobase 8-pyrenylvinyl adenine (PVA). PVA residues in SNA can be induced to undergo intrastrand [2 + 2] photocycloaddition by 455 nm light. Effective cycloreversion of the PVA photodimer results from irradiation with 340 nm light. These reactions occurred in high yield, rapidly, selectively, and reversibly. When the PVA-SNA/RNA duplex was irradiated with 455 nm light, almost complete dissociation of the duplex was attained, and 340 nm light restored duplex formation by cycloreversion. This is the first example of use of photocycloaddition and cycloreversion to photoregulate canonical duplex formation and dissociation reversibly at constant temperature. Thus, SNA bearing PVA residues have potential for use in photocontrollable biological tools targeting endogenous RNAs in cells as well as photodriven SNA machines.

DOI： 10.1021/jacs.9b03267

cis-On/trans-Off of DNA Hybridization with Alkylthio-azobenzene on L-Threoninol Responding to Visible Light 招待有り査読有り国際共著

Asanuma H., Ishikawa T., Yamano Y., Murayama K., Liang X.

ChemPhotoChem 3 巻 ( 6 ) 頁： 418 - 424 2019年6月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemPhotoChem

An azobenzene modified with a highly branched secondary alkylthio group incorporated into DNA via an L-threoninol scaffold can serve as a photo-switch to reversibly control DNA hybridization. In the system reported here, irradiation with visible light at 400 nm causes photo-isomerization of trans-azobenzene to cis and 520 nm light causes the switch to the trans form. The DNA duplex is formed when the azobenzene is in the cis form (upon irradiation with 400 nm light) and is dissociated when the azobenzene is in the trans form (upon irradiation with 520 nm light). In our previously reported system, the switch required UV light. Here, visible light was used to induce both isomerizations, and a smooth trans-to-cis isomerization was observed below the melting temperature. This new photo-switch with visible light overcomes one of the limitations for the potential in vivo use of our method. Combining it with the previously reported photo-switch will allow the construction of elaborate DNA nanomachines.

DOI： 10.1002/cptc.201900060

Orthogonally Photocontrolled Non-Autonomous DNA Walker 査読有り国際共著

Škugor M., Valero J., Murayama K., Centola M., Asanuma H., Famulok M.

Angewandte Chemie - International Edition 58 巻 ( 21 ) 頁： 6948 - 6951 2019年5月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Angewandte Chemie - International Edition

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials, and nanofactories. Present here is an entirely light-induced DNA walker based on orthogonal photocontrol. Implementing two azobenzene derivatives, S-DM-Azo and DM-Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non-autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back-and-forth directions as desired, and is regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non-autonomous progressive molecular motors, ultimately facilitating their application at the nanoscale.

DOI： 10.1002/anie.201901272

Isothermal double-cycle catalytic system using DNAzyme and RNase H for the highly selective one-pot detection of oligonucleotides 査読有り国際共著

An R., Kawai H., Asanuma H., Komiyama M., Liang X.

Analyst 144 巻 ( 8 ) 頁： 2773 - 2779 2019年4月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Analyst

With the use of a double-cycle system involving two catalytic reactions by RNase H and DNAzyme, the signal of oligoDNAs has been specifically amplified in an isothermal mode. The precursor of DNAzyme was introduced to the system as a ring-structured and inactivated form, which involves the 6-nt RNA portion being complementary to target oligoDNA. In the presence of target oligoDNA, the RNA portion forms a DNA/RNA hetero-duplex and is cut by RNase H. This scission converts the precursor to catalytically active DNAzyme, which in turn disconnects the molecular beacon to produce the amplified signal. Because the covalent bonds were disconnected to provide discrete structural changes in both cycles, high sensitivity and specificity are obtained, indicating the strong potential of this double catalytic cycle method for versatile applications.

DOI： 10.1039/c8an02520g

N-Benzoyl-protected Peptide Nucleic Acid (PNA) Monomers Expand the Range of Nucleobases Available for PNA-DNA Chimera

Inagaki M., Uematsu R., Mizutani T., Unabara D., Araki Y., Sakamoto S., Kashida H., Nishijima M., Asanuma H., Inoue Y., Wada T.

Chemistry Letters 48 巻 ( 4 ) 頁： 341 - 344 2019年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本化学会

We synthesized N-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.

DOI： 10.1246/cl.181048

In-stem molecular beacon targeted to a 5 0 -region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity 査読有り

CiNii Research

Miyoshi Y., Ohtsuki T., Kashida H., Asanuma H., Watanabe K.

PLoS ONE 14 巻 ( 1 ) 頁： e0211505 2019年1月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS ONE

Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNA Met (eMet) and initiator tRNA Met (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5 0 - region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

DOI： 10.1371/journal.pone.0211505

Photo-regulatable DNA isothermal amplification by template-mediated ligation 査読有り

Cheng B., Kashida H., Shimada N., Maruyama A., Asanuma H.

Chemical Communications 55 巻 ( 8 ) 頁： 1080 - 1083 2019年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

By combining azobenzene-tethered oligonucleotides as modulators and poly(l-lysine)-graft-dextran (PLL-g-Dex), a chaperone polymer, to facilitate strand displacement, we successfully developed a photo-regulatable DNA isothermal amplification method. By alternating UV and visible irradiation, linear amplification was achieved. The method enables photo-regulatability and mismatch discrimination in linear amplification of the DNA target.

DOI： 10.1039/c8cc09218d

Selective binding of nucleosides to gapped DNA duplex revealed by orientation and distance dependence of FRET 査読有り

Kashida H., Kokubo Y., Makino K., Asanuma H.

Organic and Biomolecular Chemistry 17 巻 ( 28 ) 頁： 6786 - 6789 2019年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Organic and Biomolecular Chemistry

Herein we used orientation and distance dependence of Förster resonance energy transfer (FRET) to analyze the binding of nucleosides to a gapped DNA duplex. Binding isotherms and information on the structures of the complexes were obtained by monitoring FRET between pyrene and perylene, which were introduced into the DNA through d-threoninol. FRET efficiency significantly changed upon formation of a duplex with a 1-nucleotide gap and a nucleoside. The FRET plot indicated that the complex has a double helical structure similar to a nicked duplex. Cooperative binding of two nucleosides to a duplex with a 2-nucleotide gap was also revealed using FRET. Various drug-nucleic acids interactions could be investigated using this sensitive and facile method.

DOI： 10.1039/c9ob00946a

The DNA duplex as an aqueous one-Dimensional soft crystal scaffold for photochemistry 招待有り

Asanuma H., Murayama K., Kamiya Y., Kashida H.

Bulletin of the Chemical Society of Japan 91 巻 ( 12 ) 頁： 1739 - 1748 2018年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本化学会

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 D-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 D-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.

DOI： 10.1246/bcsj.20180278

CiNii Research

Quantitative evaluation of energy migration between identical chromophores enabled by breaking symmetry 査読有り

Kashida H., Kawai H., Maruyama R., Kokubo Y., Araki Y., Wada T., Asanuma H.

Communications Chemistry 1 巻 ( 1 ) 2018年12月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Communications Chemistry

Energy migration between the identical chromophores is a necessary process in both natural and artificial photosynthesis. The distance and orientation dependence of energy migration have not been experimentally investigated in detail. Here we propose a method to investigate energy migration. Two fluorophores are introduced into one strand of a DNA duplex with a quencher placed opposite one of fluorophores. This design enables asymmetrization of identical fluorophores and allows one fluorophore to behave as an acceptor. The emission intensities and lifetimes decrease depending on the efficiency of energy migration. Distance and orientation dependence are successfully quantified, and the excitation energy migration efficiencies measured are in excellent agreement with those calculated based on Förster theory. We also demonstrate that multi-step energy migration among four fluorophores can be estimated from the theory. These results may provide a basis for design and preparation of efficient light-harvesting photonic devices and chemical probes.

DOI： 10.1038/s42004-018-0093-0

Bifacial Nucleobases for Hexaplex Formation in Aqueous Solution 査読有り

Kashida H., Hattori Y., Tazoe K., Inoue T., Nishikawa K., Ishii K., Uchiyama S., Yamashita H., Abe M., Kamiya Y., Asanuma H.

Journal of the American Chemical Society 140 巻 ( 27 ) 頁： 8456 - 8462 2018年7月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of the American Chemical Society

Although DNA can form triplex and quadruplex structures through hydrogen bonds, design and preparation of structures with more than five strands is difficult even when artificial nucleic acids are used. Herein we report a hexaplex formed by oligomers of artificial nucleic acids bearing bifacial molecules on d-threoninol. Aminopyrimidine and cyanuric acid derivatives were selected as bases because they have complementary hydrogen bonding patterns. The complex formed by aminopyrimidine and cyanuric acid decamers melted with large hysteresis. Hexaplex formation was indicated by gel electrophoresis, size exclusion chromatography and atomic force microscopy imaging, and proven directly through native mass spectrometry. CD measurements and molecular dynamics simulations indicated that the hexaplex adopts a helical structure. The hexaplex formation was highly dependent on pH and the presence of divalent cations. The hexaplex was stable in aqueous solution, and its unique structure and properties may lead to novel nanostructures, molecular assemblies, metal sensors, and ion channels.

DOI： 10.1021/jacs.8b02807

Does Treatment of Impaired Glucose Tolerance Improve Cardiovascular Outcomes in Patients with Previous Myocardial Infarction?

DNA-assisted swarm control in a biomolecular motor system 査読有り

Keya Jakia Jannat, Suzuki Ryuhei, Kabir Arif Md. Rashedul, Inoue Daisuke, Asanuma Hiroyuki, Sada Kazuki, Hess Henry, Kuzuya Akinori, Kakugo Akira

NATURE COMMUNICATIONS 9 巻頁： 453 2018年1月

増大特集細胞多様性解明に資する光技術─見て,動かす Ⅱ.見えなかったものを視る生細胞内RNAイメージングのための人工核酸

樫田啓, 浅沼浩之

生体の科学 68 巻 ( 5 ) 頁： 440 - 441 2017年10月

Asakura M., Kim J., Asanuma H., Hamasaki T., Tsukahara K., Higashino Y., Ishikawa T., Nakama Y., Koba S., Maruyama Y., Tsujimoto M., Himeno H., Ohkusa T., Fujino S., Shimizu M., Endo T., Yoda S., Muroya T., Murohara T., Ohte N., Suzuki H., Kohno T., Fukui K., Shiono T., Takase H., Uzui H., Nagai Y., Hashimoto Y., Ikeda S., Mizuno S., Tamita K., Fujita M., Satake K., Kinoshita Y., Nunohiro T., Sakagami S., Higaki J., Morii I., Sawada R., Hiasa Y., Shigemasa T., Nakahama M., Sata M., Doi O., Ueda T., Yamada T., Yamanouchi T., Yamaguchi H., Morita Y., Hayashi H., Kitakaze M.

Cardiovascular Drugs and Therapy 31 巻 ( 4 ) 頁： 401 - 411 2017年8月

記述言語：日本語出版者・発行元：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

Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in: Mdx myotubes

Le B., Murayama K., Shabanpoor F., Asanuma H., Veedu R.

RSC Advances 7 巻 ( 54 ) 頁： 34049 - 34052 2017年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：RSC Advances

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 splice-modulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20-mer 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

Development of Pseudo Base-Pairs on D-Threoninol which Exhibit Various Functions

Kashida Hiromu, Asanuma Hiroyuki

Bulletin of the Chemical Society of Japan 90 巻 ( 5 ) 頁： 475 - 484 2017年

記述言語：英語出版者・発行元：公益社団法人日本化学会

The authors have developed various kinds of pseudo base pairs using a d-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.

DOI： 10.1246/bcsj.20160371

Visible-Light-Triggered Cross-Linking of DNA Duplexes by Reversible [2+2] Photocycloaddition of Styrylpyrene

Doi T., Kawai H., Murayama K., Kashida H., Asanuma H.

Chemistry - A European Journal 22 巻 ( 30 ) 頁： 10533 - 10538 2016年7月

記述言語：日本語出版者・発行元：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

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Hasan M., Nishimoto A., Ohgita T., Hama S., Kashida H., Asanuma H., Kogure K.

Journal of Controlled Release 228 巻頁： 20 - 25 2016年4月

記述言語：日本語出版者・発行元：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

Terminus-free siRNA prepared by photo-crosslinking activated via slicing by Ago2

天然に存在しない非環状構造の核酸アナログ-人工の非環状骨格で神の設計した環状骨格に挑む

浅沼浩之

化学 71 巻頁： 2016 2016年

Kamiya Y., Iishiba K., Doi T., Tsuda K., Kashida H., Asanuma H.

Biomaterials Science 3 巻 ( 12 ) 頁： 1534 - 1538 2015年12月

記述言語：日本語出版者・発行元：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

Pre-organized Guide RNA in the Cas9 Complex Is Ready for the Selection of Target Double-Stranded DNA

Kamiya Y., Asanuma H.

ChemBioChem 16 巻 ( 16 ) 頁： 2273 - 2275 2015年11月

樫田啓, 村山恵司, 浅沼浩之

生体の科学 66 巻 ( 2 ) 頁： 145 - 150 2015年4月

Detection of DNA target with highly enhanced specificity by self-circularization rolling circle amplification

Wang X., Wang X., Dong P., Suzuki M., Asanuma H., Liang X.

Lecture Notes in Electrical Engineering 251 LNEE 巻 ( VOL. 3 ) 頁： 1449 - 1458 2014年2月

記述言語：日本語出版者・発行元：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

SNA: A nucleic acid analogue that can control the helical structure by sequence design

Asanuma H., Murayama K., Kashida H.

Kobunshi 62 巻 ( 9 ) 頁： 513 - 514 2013年9月

記述言語：日本語出版者・発行元：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.

A “sugar-deficient” G-quadruplex: Incorporation of aTNA in G4 structures

Zhou J., Murayama K., Amrane S., Rosu F., Kashida H., Bourdoncle A., Asanuma H., Mergny J.L.

Chemical Science 4 巻 ( 9 ) 頁： 3693 - 3698 2013年7月

記述言語：日本語出版者・発行元：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

P-stilbazole moieties as artificial base pairs for photo-cross-linking of DNA duplex

Kashida H., Doi T., Sakakibara T., Hayashi T., Asanuma H.

Journal of the American Chemical Society 135 巻 ( 21 ) 頁： 7960 - 7966 2013年5月

記述言語：日本語出版者・発行元：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

Quencher-free linear probe with multiple fluorophores on an acyclic scaffold

Asanuma H., Akahane M., Kondo N., Osawa T., Kato T., Kashida H.

Chemical Science 3 巻 ( 11 ) 頁： 3165 - 3169 2012年11月

記述言語：日本語出版者・発行元：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

Reversed assembly of dyes in an RNA duplex compared with those in DNA

Fujii T., Urushihara M., Kashida H., Ito H., Liang X., Yagi-Utsumi M., Kato K., Asanuma H.

Chemistry - A European Journal 18 巻 ( 42 ) 頁： 13304 - 13313 2012年10月

記述言語：日本語出版者・発行元：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

Model of elongation of short DNA sequence by thermophilic DNA polymerase under isothermal conditions

Kato T., Liang X., Asanuma H.

Biochemistry 51 巻 ( 40 ) 頁： 7846 - 7853 2012年10月

記述言語：日本語出版者・発行元：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

A photon-fueled DNA nanodevice that contains two different photoswitches

Nishioka H., Liang X., Kato T., Asanuma H.

Angewandte Chemie - International Edition 51 巻 ( 5 ) 頁： 1165 - 1168 2012年1月

記述言語：日本語出版者・発行元：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

A polycation-chaperoned in-stem molecular beacon system

Asanuma H., Osawa T., Kashida H., Fujii T., Liang X., Niwa K., Yoshida Y., Shimada N., Maruyama A.

Chemical Communications 48 巻 ( 12 ) 頁： 1760 - 1762 2012年1月

記述言語：日本語出版者・発行元：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

Nick sealing by T4 DNA ligase on a modified DNA template: Tethering a functional molecule on D -threoninol

Liang X., Fujioka K., Asanuma H.

Chemistry - A European Journal 17 巻 ( 37 ) 頁： 10388 - 10396 2011年9月

記述言語：日本語出版者・発行元：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

A cationic dye triplet as a unique "glue" that can connect fully matched termini of DNA duplexes

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

Chemistry - A European Journal 17 巻 ( 9 ) 頁： 2614 - 2622 2011年2月

記述言語：日本語出版者・発行元：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

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

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

Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 6518 LNCS 巻頁： 112 - 122 2011年2月

記述言語：日本語出版者・発行元：Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)

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

DOI： 10.1007/978-3-642-18305-8_11

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

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

Angewandte Chemie - International Edition 49 巻 ( 12 ) 頁： 2167 - 2170 2010年3月

記述言語：日本語出版者・発行元：Angewandte Chemie - International Edition

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

DOI： 10.1002/anie.200907082

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

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

FEBS Journal 277 巻 ( 6 ) 頁： 1551 - 1561 2010年3月

記述言語：日本語出版者・発行元：FEBS Journal

With the use of photoresponsive T7 promoters tethering two 2′-methylazobenzenes or 2′,6′-dimethylazobenzenes, highly efficient photoregulation of DNA transcription was obtained. After UV-A light irradiation (320-400 nm), the rate of transcription with T7 RNA polymerase and a photoresponsive promoter involving two 2′,6′-dimethylazobenzenes was 10-fold faster than that after visible light irradiation (400-600 nm). By attaching a nonmodified azobenzene and 2′,6′-dimethylazobenzene at the two positions, respectively, and by utilizing the different cis→trans thermal stability between cis-nonmodified azobenzene and cis-2′,6′- dimethylazobenzene, four species of T7 promoter (cis-cis, trans-cis, cis-trans, and trans-trans) were obtained. The four species showed transcriptional activity in the order of cis-cis > cis-trans > trans-cis > trans-trans. Kinetic analysis revealed that the Km for the cis-cis promoter (both of the introduced azobenzene derivatives were in the cis form) and T7 RNA polymerase was 68 times lower than that for the trans-trans form, indicating that high photoregulatory efficiency was mainly due to a remarkable difference in affinity for RNA polymerase. The present approach is promising for the creation of biological tools for artificially controlling gene expression, and as a photocontrolled system for supplying RNA fuel for RNA-powered molecular nanomachines. © 2010 FEBS.

DOI： 10.1111/j.1742-4658.2010.07583.x

Molecular imprinting of cyclodextrin to physiologically active oligopeptides in water

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

Supramolecular Chemistry 22 巻 ( 3 ) 頁： 149 - 155 2010年3月

記述言語：日本語出版者・発行元：Supramolecular Chemistry

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

DOI： 10.1080/10610270902980622

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

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

Chemistry - A European Journal 16 巻 ( 8 ) 頁： 2479 - 2486 2010年2月

記述言語：日本語出版者・発行元：Chemistry - A European Journal

To increase the apparent Stokes' shift of perylene, pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to achieve the efficient fluorescence resonance energy transfer (FRET) from pyrene to perylene. Multiple donors were introduced in the vicinity of acceptors through d-threoninol and natural base pairs were inserted between the dyes. Accordingly, donors and acceptors could be accumulated inside the DNA without forming an undesired excimer/ exciplex. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 460 nm was observed from perylene when excited at 345 nm at which pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high apparent FRET efficiency (Fφ1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Fçrster radius (R0) of the donor pyrene. Next, this FRET system was used to enlarge the Stokes' shift of the DNA probe, which can discriminate a one-base deletion mutant from wild type with a model system by incorporation of multiple donors into DNA. Two perylene moieties were tethered to the DNA on both sides of the intervening base, and two pyrenes were further inserted in the vicinity of the perylenes as an antenna. Hybridization of this FRET probe with a fully matched DNA allowed monomer emission of perylene when the pyrenes were excited. In contrast, excimer emission was generated by hybridization with a one-base deletion mutant. Thus, the apparent Stokes' shift was enhanced without loss of efficiency in the detection of the deletion mutant. © 2010 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.

DOI： 10.1002/chem.200902078

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

Nishioka H., Liang X., Asanuma H.

Chemistry - A European Journal 16 巻 ( 7 ) 頁： 2054 - 2062 2010年2月

記述言語：日本語出版者・発行元：Chemistry - A European Journal

We synthesized various azobenzenes methylated at their ortho positions with respect to the azo bond for more effective photoregulation of DNA hybridization. Photoregulatory efficiency, evaluated from the change of T m (ΔTm) induced by trans-cis isomerization, was significantly improved for all ortho-modified azobenzenes compared with non-modified azobenzene due to the more stabilized trans form and the more destabilized cis form. Among the synthesized azobenzenes, 4-carboxy-2',6'- dimethylazobenzene (2',6'-Me-Azo), in which two ortho positions of the distal benzene ring with respect to carboxyl group were methylated, exhibited the largest ΔTm, whereas the newly synthesized 2,6-Me-Azo (4-carboxy-2,6- dimethylazobenzene), which possesses two methyl groups on the two ortho positions of the other benzene ring, showed moderate improvement of ΔTm. Both NMR spectroscopic analysis and computer modeling revealed that the two methyl groups on 2',6'-Me-Azo were located near the imino protons of adjacent base pairs; these stabilized the DNA duplex by stacking interactions in the trans form and destabilized the DNA duplex by steric hindrance in the cis form. In addition, the thermal stability of cis-2',6'-Me-Azo was also greatly improved, but not that of cis2,6-Me-Azo. Solvent effects on the half-life of the cis form demonstrated that cis-to-trans isomerization of all the modified azobenzenes proceeded through an inversion route. Improved thermal stability of 2',6'-Me-Azo but not 2,6-Me-Azo in the eis form was attributed to the retardation of the inversion process due to steric hindrance between lone pair electrons of the π orbital of the nitrogen atom and the methyl group on the distal benzene ring. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/chem.200902789

Femtosecond photoisomerization study on azobenzene-derivative bound by DNA

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

Optics InfoBase Conference Papers 2010年

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

Fujii T., Kashida H., Asanuma H.

Chemistry - A European Journal 15 巻 ( 39 ) 頁： 10092 - 10102 2009年10月

記述言語：日本語出版者・発行元：Chemistry - A European Journal

To test the molecular exciton theory for heterodimeric chromophores, various heterodimers and clusters, in which two different dyes were stacked alternately, were prepared by hybridizing two oligodeoxyribonucleotides (ODNs), each of which tethered a different dye on D-threoninol at the center of the strand. NMR analyses revealed that two different dyes from each strand were stacked antiparallel to each other in the duplex, and were located adjacent to the 5'-side of a natural nucleobase. The spectroscopic behavior of these heterodimers was systematically examined as a function of the difference in the wavelength of the dye absorption maxima (Δλmax). We found that the absorption spectrum of the heterodimer was significantly different from that of the simple sum of each monomeric dye in the single Strand. When azobenzene and Methyl Red, which have λmax at 336 and 480 nm, respectively, in the single strand (Δλmax= 144nm), were assembled on ODNs, the band derived from azobenzene exhibited a small hyperchromism, whereas the band from Methyl Red showed hypochromism and both bands shifted to a longer wavelength (bathochromism). These hyper- and hypochromisms were further enhanced in a heterodimer derived from 4'-methylthioazobenzene and Methyl Red, which had a much smaller Δλmax (82 nm; λmax = 398 and 480 nm in the single-strand, respectively). With a combination of 4'-dimethyl-amino-2- nitroazobenzene and Methyl Red, which had an even smaller Δλ max (33 nm), a single sharp absorption band that was apparently different from the sum of the single-stranded spectra was observed. These changes in the intensity of the absorption band could be explained by the molecular exciton theory, which has been mainly applied to the spectral behavior of H- and/or Jaggregates composed of homo dyes. However, the bathochromic band shifts observed at shorter wavelengths did not agree with the hypsochromism predicted by the theory. Thus, these data experimentally verify the molecular exciton theory of heterodimerization. This coherent coupling among the heterodimers could also partly explain the bathochromicity and hypochromicity that were observed when the dyes were intercalated into the duplex. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/chem.200900962

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

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

Angewandte Chemie - International Edition 48 巻 ( 38 ) 頁： 7044 - 7047 2009年9月

記述言語：日本語出版者・発行元：Angewandte Chemie - International Edition

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

DOI： 10.1002/anie.200902367

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

Liang X., Mochizuki T., Asanuma H.

Small 5 巻 ( 15 ) 頁： 1761 - 1768 2009年8月

記述言語：日本語出版者・発行元：Small

A supra-photoswitch is designed for complete ON/OFF switching of DNA hybridization by light irradiation for the purpose of using DNA as a material for building nanostructures. Azobenzenes, attached to D-threoninols that function as scaffolds, are introduced into each DNA strand after every two natural nucleotides (in the form (NNX)n whereNandXrepresent the natural nucleotide and the azobenzene moiety, respectively). Hybridization of these two modified strands forms a supra-photoswitch consisting of alternating natural base pairs and azobenzene moieties. In this newly designed sequence, each base pair is sandwiched between two azobenzene moieties and all the azobenzene moieties are separated by base pairs. When the duplex is irradiated by visible light, the azobenzene moieties take the trans form and this duplex is surprisingly stable compared to the corresponding native duplex composed of only natural oligonucleotides. On the other hand, when the azobenzene moieties are isomerized to the cis form by UV light irradiation, the duplex is completely dissociated. Based on this design, a DNA hairpin structure is synthesized that should be closed by visible light irradiation and opened by UV light irradiation at the level of a single molecule. Indeed, perfect ON/OFF photoregulation is attained. This is a promising strategy for the design of supra-photoswitches such as photoresponsive sticky ends on DNA nanodevices and other nanostructures. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/smll.200900223

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

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

Journal of the American Chemical Society 131 巻 ( 29 ) 頁： 9928 - 9930 2009年7月

記述言語：日本語出版者・発行元：Journal of the American Chemical Society

(Figure Presented) "Base pairs" of cationic dyes (p-methylstilbazole) were incorporated into oligodeoxyribonucleotides (ODNs). This "base pair" greatly stabilized the duplex through electrostatic and stacking interactions. The melting temperature of modified ODN was higher than those of neutral dyes and native base pairs. Further stabilization of the duplex was observed when the number of cationic dyes increased. © 2009 American Chemical Society.

DOI： 10.1021/ja9013002

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

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

Bioconjugate Chemistry 20 巻 ( 2 ) 頁： 258 - 265 2009年2月

記述言語：日本語出版者・発行元：Bioconjugate Chemistry

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

DOI： 10.1021/bc800335h

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

Kashida H., Liang X., Asanuma H.

Current Organic Chemistry 13 巻 ( 11 ) 頁： 1065 - 1084 2009年

記述言語：日本語出版者・発行元：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

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

名古屋コンファレンス報告

浅沼浩之, 早川芳宏

化学と工業 = Chemistry and chemical industry 60 巻 ( 4 ) 頁： 456 2007年4月

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

Chemical Communications ( 42 ) 頁： 4354 - 4356 2007年

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

Liang X., Kato T., Asanuma H.

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

記述言語：英語出版者・発行元：Nucleic acids symposium series (2004)

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

DOI： 10.1093/nass/nrm176

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

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

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：Polymer Preprints, Japan

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

Preparation of active DNA enzymes by introduction of intercalator

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

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：Polymer Preprints, Japan

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

Synthesis of peptide nucleic acid conjugated with carbohydrate

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

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：Polymer Preprints, Japan

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

Synthesis of fluorescent DNA probe by use of dye polarization

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

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：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.

Stabilization of DNA duplex by introducing intercalators into DNA through D-threoninol

Kashida H., Komiyama M., Asanuma H.

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：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.

Recognition of bioactive oligopeptide by imprinted cyclodextrin polymer in water

Sumaoka J., Shirasaka K., Katayama M., Song S., Nagaoka S., Asanuma H., Komiyama M.

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：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.

Preparation of new dye supramolecule by the spontaneous aggregation

Yamada A., Kashida H., Komiyama M., Asanuma H.

Polymer Preprints, Japan 55 巻 ( 1 ) 2006年10月

記述言語：日本語出版者・発行元：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.

Importance of the position of vinyl group on β-cyclodextrin for the effective imprinting of amino acid derivatives and oligopeptides in water

Osawa T., Shirasaka K., Matsui T., Yoshihara S., Akiyama T., Hishiya T., Asanuma H., Komiyama M.

Macromolecules 39 巻 ( 7 ) 頁： 2460 - 2466 2006年4月

記述言語：日本語出版者・発行元：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

Photo- and thermoregulation of DNA nanomachines

Takahashi K., Yaegashi S., Asanuma H., Hagiya M.

Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) 3892 LNCS 巻頁： 336 - 346 2006年

記述言語：日本語出版者・発行元：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

Functionalization of DMA by using DNA-pyrene conjugates

Kashida H., Komiyama M., Asanuma H.

Polymer Preprints, Japan 54 巻 ( 2 ) 頁： 5092 - 5093 2005年12月

記述言語：日本語出版者・発行元：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.

Acceleration of influenza virus clearance by Th1 cells in the nasal site of mice immunized intranasally with adjuvant-combined recombinant nucleoprotein

Photo- and Thermoregulation of DNA Nanomachines

浅沼浩之, 萩谷昌己

DNA11, Eleventh International Meeting on DNA Based Computers, Preliminary Proceedings 頁： 147 - 156 2005年

バイオナノデバイスとしての人工DNA 査読有り

浅沼浩之

化学と教育 53 頁： 12 - 15 2005年

NMR study on the photoresponsive DNA tethering an azobenzene. Assignment of the absolute configuration of two diastereomers and structure determination of their duplexes in the trans-form.

Liang X, Asanuma H, Kashida H, Takasu A, Sakamoto T, Kawai G, Komiyama M

Journal of the American Chemical Society 125 巻 ( 52 ) 頁： 16408 - 15 2003年12月

DNA-Naphthyl Red conjugate as a visualizing probe of DNA hybridization.

Asanuma H, Kashida H, Liang X, Komiyama M

Chemical communications (Cambridge, England) ( 13 ) 頁： 1536 - 7 2003年7月

DNA-dye conjugates for controllable H aggregation(1).

Asanuma H, Shirasuka K, Takarada T, Kashida H, Komiyama M

Journal of the American Chemical Society 125 巻 ( 8 ) 頁： 2217 - 23 2003年2月

Development of a probe DNA which accompanies color change on hybridization.

Kashida H, Asanuma H, Komiyama M

Nucleic acids research. Supplement (2001) ( 3 ) 頁： 143 - 4 2003年

Effective photo-regulation of transcription reaction by SP6 RNA polymerase with modified DNA tethering multiple azobenzenes.

Liu M, Asanuma H, Komiyama M

Nucleic acids research. Supplement (2001) ( 3 ) 頁： 265 - 6 2003年

Photo-regulation of DNA function by azobenzene-tethered oligonucleotides.

Asanuma H, Matsunaga D, Liu M, Liang X, Jhao J, Komiyama M

Nucleic acids research. Supplement (2001) ( 3 ) 頁： 117 - 8 2003年

Photoregulation of the transcription reaction of T7 RNA polymerase by tethering an azobenzene to the promoter.

Asanuma H, Tamaru D, Yamazawa A, Liu M, Komiyama M

Chembiochem : a European journal of chemical biology 3 巻 ( 8 ) 頁： 786 - 9 2002年8月

Photoregulation of DNA triplex formation by azobenzene.

Liang X, Asanuma H, Komiyama M

Journal of the American Chemical Society 124 巻 ( 9 ) 頁： 1877 - 83 2002年3月

Spectroscopic anatomy of molecular-imprinting of cyclodextrin. Evidence for preferential formation of ordered cyclodextrin assemblies.

Hishiya T, Asanuma H, Komiyama M

Journal of the American Chemical Society 124 巻 ( 4 ) 頁： 570 - 5 2002年1月

Photo-regulation of transcription by RNA polymerase with azobenzene-tethered promoter.

Asanuma H, Liu M, Tamaru D, Liang X, Komiyama M

Nucleic acids research. Supplement (2001) ( 2 ) 頁： 75 - 6 2002年

Enantioselective Incorporation of Azobenzenes into Oligodeoxyribonucleotide for Effective Photoregulation of Duplex Formation This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Molecular Synchronization for Design of New Materials System). The support by the Grant from "Research for the Future" Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00301) is also acknowledged.

Asanuma H, Takarada T, Yoshida T, Tamaru D, Liang X, Komiyama M

Angewandte Chemie (International ed. in English) 40 巻 ( 14 ) 頁： 2671 - 2673 2001年7月

Enantioselective Incorporation of Azobenzenes into Oligodeoxyribonucleotide for Effective Photoregulation of Duplex Formation.

Asanuma H, Takarada T, Yoshida T, Tamaru D, Liang X, Komiyama M

Angewandte Chemie (International ed. in English) 40 巻 ( 14 ) 頁： 2671 - 2673 2001年7月

Tamura S.I., Miyata K., Matsuo K., Asanuma H., Takahashi H., Nakajima K., Suzuki Y., Aizawa C., Kurata T.

Journal of Immunology 156 巻 ( 10 ) 頁： 3892 - 3900 1996年5月

記述言語：日本語出版者・発行元：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.

Pseudo base pairs that exhibit high duplex stability and orthogonality through covalent and non─covalent interactions 招待有り

Reversible Optical Control of Receptor Tyrosine Kinase Activity and ERK Dynamics Using Azobenzene-Carrying DNA Aptamer Agonist 査読有り

Wakano, M.; Tsunoda, M.; Murayama, K.; Morimoto, J.; Ueki, R.; Aoyama-Ishiwatari, S.; Hirabayashi, Y.; Asanuma, H.; Sando, S.

J. Am. Chem. Soc. 147 巻 2025年3月

Protocol for controlling the strand selectivity of siRNA using acyclic artificial nucleic acids 招待有り査読有り

Current protocols 5 巻頁： e70103 2025年3月

Reaction and interaction dynamics of azobenzene-tethered DNA with T7 RNA polymerase 査読有り

Takekawa, G.; Nakasone, Y.; Kamiya, Y.; Asanuma, H.; Terazima, M.

Phys. Chem. Chem. Phys. 27 巻頁： 3302 - 3312 2025年1月

Localized Control of the Swarming of Kinesin-Driven Microtubules Using Light 査読有り国際共著

Akter, M.; Kabir, A.M.R.; Keya, J.J.; Sada, K.; Asanuma, H.; Kakugo, A.

ACS Omega 9 巻頁： 377348 - 37753 2024年8月

Photosensitization of TiO2 Electrodes Immobilized with Chiral Plasmonic Au Nanocolloids by Circularly Polarized Light Irradiation 招待有り査読有り

Kameyama, T.; Isogawa, S.; Akiyoshi, K.; Kashida, H.; Asanuma, H.; Torimoto, T.

The Journal of Chemical Physics 160 巻頁： 094709 2024年3月

DNA reaction system that acquires classical conditioning 招待有り査読有り

ACS Synthetic Biology 頁： 521 - 529 2024年1月

Highly Functional Acyclic Xeno Nucleic Acids 招待有り査読有り

Murayama, K.; Okita, H.; Asanuma, H.

Bull. Chem. Soc. Jpn. 96 巻頁： 1179 - 1187 2023年11月

Illuminating miRNA inhibition: Visualizing the interaction between anti-miRNA oligonucleotide and target miRNA using FRET 査読有り

Zhu, H.; Kamiya, Y.; Asanuma, H.

ACS Chemical Biology 18 巻頁： 2281 - 2289 2023年10月

Functionalization of Acyclic Xenonucleic Acid with Modified Nucleobases 招待有り査読有り

Murayama, K.; Yamano, Y.; Asanuma, H.

Polym. J. 55 巻頁： 743 - 752 2023年4月

Orientational control of circularly polarized luminescence from pyrene clusters using a DNA scaffold 査読有り

Chemistry - A European Journal 2023年2月

Orientational control of circularly polarized luminescence from pyrene clusters using a DNA scaffold 査読有り

Kashida, H.; Ito, Y.; Kakuta, T.; Ogoshi, T.; Asanuma, H.

Chemistry - A European Journal 2023年2月

DNA二重鎖を利用したFRET配向依存性の検討招待有り査読有り

樫田　啓, 浅沼浩之

光化学 53 巻 ( 3 ) 頁： 148 - 153 2022年12月

DNA Concentration Regulator That can be Driven for a Long Time 査読有り

Nakakuki, T.; Murayama, K.; Asanuma, H.

New Gener. Comput. 40 巻頁： 681 - 702 2022年9月

RNA以前の世界を妄想する招待有り査読有り

浅沼浩之, 村山恵司

化学と工業 75 巻 ( 8 ) 頁： 552 - 554 2022年8月

Kinesin motors driven microtubule swarming triggered by UV light 査読有り

Ishii, S.; Akter, M.; Murayama, K.; Kabir, A. M. R.; Asanuma, H.; Sada, K.; Kakugo, A.

Polym. J. 54 巻頁： 1501 - 1507 2022年7月

Methyl group configuration on acyclic threoninol nucleic acids (aTNAs) impacts supramolecular properties 査読有り

Murayama, K.; Kashida, H.; Asanuma, H.

Org. Biomol. Chem. 20 巻頁： 4115 - 4122 2022年3月

Renewable DNA proportional-integral controller with photoresponsive molecules 招待有り査読有り

Tamba, M.; Murayama, K.; Asanuma, H.; Nakakuki, T.

Micromachines 13 巻頁： 193 2022年2月

Signal Amplification Circuit Composed of Serinol Nucleic Acid for RNA Detection 査読有り

Chen, Y.; Murayama, K.; Asanuma, H.

Chemistry Letters 51 巻頁： 330 - 333 2022年2月

Unexpected Dissociation of Photoresponsive UV-ON DNA Carrying p-tert-Butyl Azobenzene under UV Light Irradiation 査読有り

Ishii, S.; Murayama, K.; Sada, K.; Asanuma, H.; Kakugo, A.

Chem. Lett. 51 巻頁： 292 - 295 2022年1月

Signal Amplification Circuit Composed of Serinol Nucleic Acid for RNA Detection 査読有り

Chen, Y.; Murayama, K.; Asanuma, H.

Chem. Lett. 51 巻頁： 330 - 333 2022年1月

Renewable DNA proportional-integral controller with photoresponsive molecules 招待有り査読有り

Tamba, M.; Murayama, K.; Asanuma, H.; Nakakuki, T.

Micromachines 13 巻頁： 193 2022年1月

Unexpected Dissociation of Photoresponsive UV-ON DNA Carrying p-tert-Butyl Azobenzene under UV Light Irradiation 査読有り

Ishii, S.; Murayama, K.; Sada, K.; Asanuma, H.; Kakugo, A.

Chemistry Letters 51 巻頁： 292 - 295 2022年1月

樫田啓, 浅沼浩之

有機合成化学協会誌 79 巻 ( 11 ) 頁： 1013 - 1019 2021年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人有機合成化学協会

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.

DOI： 10.5059/yukigoseikyokaishi.79.1013

CiNii Research

光駆動型DNAナノマシン招待有り査読有り

浅沼浩之, 村山恵司, 神谷由紀子, 樫田　啓

高分子 70 巻頁： 550 - 552 2021年10月

Preparation of Artificial Hexaplex Composed of Non-Natural Nucleic Acid 招待有り査読有り

Makino K., Hattori Y., Kashida H., Asanuma H.

Current Protocols 1 巻 ( 4 ) 頁： e106 2021年4月

記述言語：日本語出版者・発行元：Current Protocols

This article describes synthetic procedures for acyclic D-threoninol nucleic acid tethering of bifacial nucleobases. Because these nucleobases have complementary hydrogen bonding sites on both sides, their oligomers can form a hexaplex. These hexaplexes are suitable for use as metal or pH sensors and as supramolecular motifs. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of D-threoninol backbone. Basic Protocol 2: Synthesis of D-aTNA tethering cyanuric acid. Basic Protocol 3: Synthesis of D-aTNA tethering a 2-aminopyrimidine moiety. Basic Protocol 4: Synthesis of D-aTNA tethering a 2,4,6-triaminopyrimidine moiety. Basic Protocol 5: Synthesis of D-aTNA oligomer tethering cyanuric acid, 2-aminopyrimidine, or 2,4,6-triaminopyrimidine.

DOI： 10.1002/cpz1.106

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA 査読有り

Nanotechnology 2021年3月

Development of pre-miRNAs modification with FRET dye pair for intracellular visualization of processing intermediates generated in cells 査読有り

Kamiya, Y.; Kamimoto, H.; Zhu, H. ; Asanuma, H.

Sensors 21 巻頁： 1785 2021年3月

Development and modification of pre-mirnas with a fret dye pair for the intracellular visualization of processing intermediates that are generated in cells 招待有り査読有り

Kamiya Y., Kamimoto H., Zhu H., Asanuma H.

Sensors 21 巻 ( 5 ) 頁： 1 - 14 2021年3月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

DNA二重鎖の高い安定性と直交性をもつ疑似塩基対の開発

Pyrene modified serinol nucleic acid nanostructure converts chirality of threoninol nucleic acids into circularly polarized luminescence signals 招待有り査読有り

Kashida, H.; Nishikawa, K.; Ito Y.; Murayama, K.; Hayashi, I.; Kakuta, T.; Ogoshi, T.; Asanuma, H.

2021年1月

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA 査読有り

Takahashi, H.; Yasui, T.; Kashida, H.; Makino, K.; Shinjo, K.; Liu, Q.; Shimada, T.; Rahong, S.; Kaji, N.; Asanuma, H.; Baba, Y.

Nanotechnology 32 巻頁： 255301 2021年

核酸医薬の創製に向けた人工核酸の開発招待有り査読有り

樫田啓、村山恵司、浅沼浩之

バイオマテリアル 38 巻 ( 2 ) 頁： 124 - 129 2020年4月

The Use of Serinol Nucleic Acids as Ultrasensitive Molecular Beacons 招待有り査読有り

Murayama Keiji, Kashida Hiromu, Asanuma Hiroyuki

NON-NATURAL NUCLEIC ACIDS: METHODS AND PROTOCOLS 1973 巻頁： 261-279 2019年

Development of Visible-Light-Responsive RNA Scissors Based on a 10-23 DNAzyme 査読有り

Kamiya Yukiko, Arimura Yu, Ooi Hideaki, Kato Kenjiro, Liang Xing-Guo, Asanuma Hiroyuki

CHEMBIOCHEM 19 巻 ( 12 ) 頁： 1305 - 1311 2018年6月

DNA Microcapsule for Photo-Triggered Drug Release Systems

Kamiya Yukiko, Yamada Yoshinobu, Muro Takahiro, Matsuura Kazunori, Asanuma Hiroyuki

CHEMMEDCHEM 12 巻 ( 24 ) 頁： 2016-2021 2017年12月

Introduction of 2,6-Diaminopurines into Serinol Nucleic Acid Improves Anti-miRNA Performance 査読有り

Kamiya Yukiko, Donoshita Yuka, Kamimoto Hiroshi, Murayama Keiji, Ariyoshi Jumpei, Asanuma Hiroyuki

CHEMBIOCHEM 18 巻 ( 19 ) 頁： 1917-1922 2017年10月

Chaperone-Polymer-Assisted, Photodriven DNA Strand Displacement

Cheng Bohao, Kashida Hiromu, Shimada Naohiko, Maruyama Atsushi, Asanuma Hiroyuki

CHEMBIOCHEM 18 巻 ( 16 ) 頁： 1568-1572 2017年8月

D-aTNA Circuit Orthogonal to DNA Can Be Operated by RNA Input via SNA

Murayama Keiji, Nagao Ryuya, Asanuma Hiroyuki

CHEMISTRYSELECT 2 巻 ( 20 ) 頁： 5624-5627 2017年7月

Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in mdx myotubes 査読有り

Le, B. T.; Murayama, K.; Shabanpoor, F.; Asanuma, H.; Veedu, R. N.

RSC Advances 7 巻頁： 34049-34052 2017年7月

光応答性DNAの開発―ナノ環境を汚染しない分子マシンへの応用― 招待有り

浅沼浩之

現代化学 556 巻 ( 7 ) 頁： 50-55 2017年7月

DNA二重鎖の高い安定性と直交性をもつ疑似塩基対の開発招待有り査読有り

樫田啓、浅沼浩之

高分子論文集 74 巻 ( 4 ) 頁： 257-264 2017年7月

Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes

Kashida Hiromu, Kurihara Ayako, Kawai Hayato, Asanuma Hiroyuki

NUCLEIC ACIDS RESEARCH 45 巻 ( 11 ) 2017年6月

Development of pseudo base-pairs on D-threoninol which exhibit various functions 招待有り査読有り

Kashida, H.; Asanuma, H.

Bull. Chem. Soc. Jpn. 90 巻頁： 475 2017年5月

Design of photo-functional oligonucleotide by "copolymerization" of natural nucleobases with base-surrogate prepared from acyclic scaffold 招待有り査読有り

Asanuma, H.; Murayama, K.; Kamiya, Y.; Kashida, H.

Polymer J. 49 巻頁： 279-289 2017年3月

DNA二重鎖による色素会合体の精密位置配向制御と光化学への展開招待有り査読有り

浅沼浩之、樫田啓

固体物理 52 巻 ( 3 ) 頁： 149-158 2017年3月

Effect of Methyl Group on Acyclic Serinol Scaffold for Tethering Dyes on the DNA Duplex Stability

Murayama Keiji, Asanuma Hiroyuki

CHEMBIOCHEM 18 巻 ( 1 ) 頁： 142-149 2017年1月

Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes 査読有り

Kashida, H.; Kurihara, A.; Kawai, H.; Asanuma, H.

Nucleic Acids Res. 45 巻 ( 11 ) 頁： e105 2017年

樫田啓, 浅沼浩之

高分子論文集 74 巻 ( 4 ) 頁： 257 - 264 2017年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人高分子学会

天然核酸塩基は高い二重鎖安定性と直交性という化学的に魅力的な性質をもつ．この天然塩基対に代わる新たな塩基対を開発することができれば，天然がもちえない機能をもった核酸材料の開発が可能となる．筆者らは非環状骨格であるD-threoninolに着目し，これを介した疑似塩基対の開発を行ってきた．また，疑似塩基対の化学構造が天然塩基対とまったく異なるにもかかわらず二重鎖の安定化と直交性という天然塩基対の化学的機能を模倣できることを明らかにした．本報ではとくに疑似塩基対の安定性に焦点を当て，筆者らの最近の研究成果と今後の展望について概説する．

DOI： 10.1295/koron.2017-0009