研究者詳細 - 浅沼　浩之

論文 - 浅沼　浩之

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

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

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

DOI： 10.1246/bcsj.20160371

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

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

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

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

特集使える最新ケミカルバイオロジー B.核酸のケミカルバイオロジー新規人工核酸SNAを用いたRNAイメージング

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

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