研究者詳細 - 浅沼　浩之

論文 - 浅沼　浩之

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

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 <sup>0</sup> -region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity 査読有り

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

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

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

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