Studies on stereoregulated phosphorothioates for their gene-regulatory ability

立体调控硫代磷酸酯基因调控能力的研究

• 批准号: 08458176
• 负责人: MAKINO Keisuke
• 金额: $ 0.96万
• 依托单位: KYOTO UNIVERSITY (1997-1998)Kyoto Institute of Technology (1996)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458176/
• 关键词: Antisense technique; Phosphorothiate DNA; Local structure analysis for hybrids; Effect of thiophosphate stereoisomerism; Nonspecific antiviral activity; Structure of telomere C-repeat; Four-stranded structure; Stereoselective synthesis; 核磁気共鳴法; 分

In the antisense technique, one of the most promising diagnostic methods for severe viral diseases, . phosphorothioate oligodeoxynucleotides (S-oligos) which has a thiophosphate linkage have been investigated exclusively. To apply this method in clinic, however, there has been a crucial unsolved problem that one should identify and prepare the truly effective specific structure among numbers of stereoisomeric S-oligos arising from the stereoisomerism of each thiophosphate linkage, and in the present study, therefore, we have performed following investigation s to overcome the above problem. (1) We have carried out 2D-NMR analysis combined with molecular dynamic calculation on the relationship between the thiophosphate Rp and Sp configurations and the S-oligo RNA/DNA duplex structure. It has been elucidated that the structure of the duplexes with RNA is similar with each other as well as with that of wild type DNA-RNA duplex, although Rp linkage produced higher Tm, indicative that the d … More uplex stability is dependent such a factor other than the duplex structure, possibly as extent of hydration. On the other hand, difference of the duplex structure was found to be responsible for S-oligo-DNA hybrid stability. These new findings indicate that the, suitable configuration depends on the target, and that the mechanism of this rule also on it. (2). Nonspecific antiviral activity of S-oligo polyC has also been studied, and found to be due to its unique four-stranded solution structure, i-motif, whose stability is dependent on its Rp or Sp configuration. Also, a possible target gene for S-oligo polyC, telomere C-repeat, has been investigated in terms of the structure, and it has been found that there are isomeric C repeat tetrads. This new discovery may give suggestion not only to understand whole mechanism for nonspecific activity of S-oligo but biological functions of telomere topologies in relation to cancer and aging. (3) Stereoselective synthesis of S-oligos, another unsolved essential subject, has been studied and use of m-chlorocresol as a leaving group in the phosphite method was found to lead to high stereoselectivity and high yield of transesterification in the basic conditions. Less

在反义技术中，这是严重病毒性疾病最有前途的诊断方法之一。已经专门研究了具有硫代磷酸酯键的硫代磷酸酯寡脱氧核苷酸（S-寡核苷酸）。然而，为了将这种方法应用于临床，一直存在一个关键的未解决的问题，即人们应该在由每个硫代磷酸酯键的立体异构产生的许多立体异构的S-寡核苷酸中鉴定和制备真正有效的特定结构，因此，在本研究中，我们进行了以下研究以克服上述问题。(1)我们用2D-NMR分析结合分子动力学计算研究了硫代磷酸酯的Rp和Sp构型与S-oligo RNA/DNA双链体结构之间的关系。已阐明，与RNA的双链体的结构彼此相似，并且与野生型DNA-RNA双链体的结构相似，尽管Rp连接产生更高的Tm，这表明与野生型DNA-RNA双链体的结构相似。 ...更多信息 双链体稳定性取决于除双链体结构以外的因素，可能是水合程度。另一方面，双链体结构的差异被认为是负责S-oligo-DNA杂交稳定性。这些新的发现表明，适当的配置取决于目标，这一规则的机制也取决于它。还研究了S-oligo polyC的非特异性抗病毒活性，发现这是由于其独特的四链溶液结构，i-基序，其稳定性取决于其Rp或Sp构型。此外，一个可能的目标基因的S-寡聚C，端粒C-重复，已被调查的结构方面，它已被发现，有异构的C重复四分体。这一新发现不仅有助于理解S-oligo非特异性活性的整个机制，而且有助于理解端粒拓扑结构与癌症和衰老相关的生物学功能。(3)研究了另一个尚未解决的重要课题S-寡聚物的立体选择性合成，发现在亚磷酸酯法中使用间氯甲酚作为离去基团导致在碱性条件下的高立体选择性和高酯交换产率。少

项目成果

K.Kanaori, K.Yokoyama, K.Tajima, N.Yamamoto, T.Okamoto, and K.Makono: "Interaction of pyrylium dye with self-complementary DNA oligomer as studied by ^1H NMR spectroscopy" Nucleosides & Nucleotides. 17. 603-611 (1998)

K.Kanaori、K.Yokoyama、K.Tajima、N.Yamamoto、T.Okamoto 和 K.Makono：“通过 ^1H NMR 光谱研究吡喃鎓染料与自互补 DNA 寡聚物的相互作用”

K.Makino et al.(7名): "Pterin-6-aldehyde,an Inhibitor of Xanthine Oxidase,Has Superoxide Anion Radical Scavenging Activity" Biochemical and Biophysical Research Communications. 233. 447-450 (1997)

K. Makino 等人（7 人）：“蝶呤-6-醛，一种黄嘌呤氧化酶抑制剂，具有超氧化物阴离子自由基清除活性”《生物化学和生物物理研究通讯》233. 447-450 (1997)。

N.Miyano-Kurosaki: "Inhibitiion of HTLV-1 induction and virus-induced syncytia formation by oligodeoxynucleotides." Virus Genes. 12:3. 205-217 (1996)

N.Miyano-Kurosaki：“通过寡脱氧核苷酸抑制 HTLV-1 诱导和病毒诱导的合胞体形成。”

T.Morii et al.: "Cooperative oligomerization enhances sequence-selective DNA binding by a short peptid" J.Am.Chem.Soc.118. 10011-10017 (1996)

T.Morii 等人：“协同寡聚化增强了短肽的序列选择性 DNA 结合”J.Am.Chem.Soc.118。

H.Kanehara, M.Mizuguchi, and K.Makino: "Isolation of oligodeoxynucleoside phosphorothioate diastereomers by the combination of DEAE ion-ex-change and reversed-phase chromatography" Nucleosides & Nucleotides. 15. 407-417 (1996)

H.Kanehara、M.Mizuguchi 和 K.Makino：“通过结合 DEAE 离子交换和反相色谱法分离寡脱氧核苷硫代磷酸酯非对映异构体”