Study to develop novel genomic drugs by using superfunctional oligonucleotide analogues

利用超功能寡核苷酸类似物开发新型基因组药物的研究

• 批准号: 12557201
• 负责人: IMANISHI Takeshi
• 金额: $ 7.81万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557201/
• 关键词: Sugar conformation; Nucleoside analogue; Oligonucleotide analogue; Artificial nucleic acid; Synthesis; Antisense; Antigene; BNA; 酵素耐性; ホスホロアミダート結合

Antisense and antigene strategies are new methodology for genomic drugs discovery. An antisense oligonucleotide hybridizes with a target mRNA and inhibits gene expression sequence-selectively in a living cell. On the other hand, an antigene oligonucleotide (triplex-forming oligonucleotides, TFOs) binds with genomic dsDNA to result in gene expression control. We previously developed various kinds of conformationally locked nucleic acids (Bridged Nucleic Aclds, BNAs). Ologonucleotides modified with BNA monomers were widely used to evaluate physical and biological properties. These results are summarized below.1) TFOs containing the 2',4'-BNA monomers were found to stabilize significantly triplex formation with the homopurine-homopyrimidine dsDNA target under near physiological conditions.2) Efficient synthetic routes for the 2',4'-BNA monomers bearing unnatural nucleobases wore developed.3) There is no natural nucleoside to recognize a CG base pair, and this psoes a serious problem in antigene strategy. A novel nucleoside analogue, 2',4'-BNA with a 2-pyridone base-unit was designed, synthesized and proved to be excellent for recognition of a CG base pair.4) Another modification was made on a phosphodiester linkage or the sugar moiety of the 2',4'-BNA oligonucleotide. The newly modified oligonucleotides showed high binding affinity with dsDNA or ssRNA target. Excellent enzymatic stability of these oligonucleotide analogues was also observed.5) Antisense 2',4'-BNA oligonucleotides inhibited expression of the target gene in living cell with high sequence-selectivity and non-cytotxicity.

反义和反基因策略是基因组药物发现的新方法。反义寡核苷酸与靶 mRNA 杂交并选择性地抑制活细胞中的基因表达序列。另一方面，反基因寡核苷酸（三链体形成寡核苷酸，TFO）与基因组 dsDNA 结合，导致基因表达控制。我们之前开发了各种构象锁定核酸（桥接核酸，BNA）。用 BNA 单体修饰的寡核苷酸被广泛用于评估物理和生物特性。这些结果总结如下。1) 含有 2',4'-BNA 单体的 TFO 在接近生理条件下可显着稳定与高嘌呤-高嘧啶 dsDNA 靶标的三链体形成。2) 2',4' 的有效合成路线-带有非天然核碱基的BNA单体出现了。3）没有天然核苷来识别CG碱基对，这给反基因策略带来了严重问题。设计、合成了一种新型核苷类似物，具有 2-吡啶酮碱基单元的 2',4'-BNA，并被证明对于 CG 碱基对的识别具有优异的性能。4) 对磷酸二酯键或糖进行了另一种修饰2',4'-BNA 寡核苷酸的部分。新修饰的寡核苷酸显示出与 dsDNA 或 ssRNA 靶标的高结合亲和力。这些寡核苷酸类似物也具有优异的酶稳定性。5)反义2',4'-BNA寡核苷酸抑制活细胞中靶基因的表达，具有高序列选择性和非细胞毒性。

项目成果

Obika, S., Hemamayi, R., Masuda, T., Sugimoto, T., Nakagawa, S., Mayumi, T., Imanishi, T.: "Inhibition of ICAM-I Gene Expression by Antisense 2', 4'-BNA Oligonucleotides"Nucleic Acids Res.Suppl.1. 145-146 (2001)

Obika, S.、Hemamayi, R.、Masuda, T.、Sugimoto, T.、Nakakawa, S.、Mayumi, T.、Imanishi, T.：“反义 2、4 抑制 ICAM-I 基因表达

Yasutoshi UCHIYAMA, Ying HUANG, Hiroshi KANAMORI, Masao UCHIDA, Takefumi DOI, Akihisa TAKAMIZAWA, Takao HAMAKUBO and Tatsuhiko KODAMA: "Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay"Hepatology Res.. 2

Yasutoshi UCHIYAMA、Ying HUANG、Hiroshi KANAMORI、Masao UCHIDA、Takefum​​i DOI、Akihisa TAKAMIZAWA、Takao HAMAKUBO 和 Tatsuhiko KODAMA：“用 C 端 21 个氨基酸截短的 NS5b 蛋白测量 HCV RdRp 活性：优化测定”Hepatology Res.. 2

Morita, K., Hasegawa, C., Kaneko, M., Tsutsumi, S., sone, J., Ishikawa, T., Imanishi, T., Koizumi, M.: "2'-O, 4'-C-Ethylene-bridged Nucleic Acids (ENA): Highly Nuclease-resistant and Thermodynamically Stable Oligonucleotides for Antisense Drug"Bioorg.Med.

森田 K.、长谷川 C.、金子 M.、堤 S.、曾根 J.、石川 T.、今西 T.、小泉 M.：“2-O、4-C

Koji MORITA, Koji YAMATE, Shin-ichi KURAKATA, Koji ABE, Takeshi IMANISHI and Makoto KOIZUMI: "Down-regulation of VEGF mRNA expression by 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) antisense oligonucleotides and investigation of non-target gene expressi

Koji MORITA、Koji YAMATE、Shin-ichi KURAKATA、Koji ABE、Takeshi IMANISHI 和 Makoto KOIZUMI：“2-O,4-C-乙烯桥核酸 (ENA) 反义寡核苷酸下调 VEGF mRNA 表达和

Uchida, M., Hino, N., Yamanaka, T., Hukushima, H., Imanishi, T., uchiyama, Y., Kodama, T., Doi, T.: "Hepatitis C virus core protein binds to a C-terminal region of NS5B RNA polymerase"Hepatology Res.. 22. 297-306 (2002)

Uchida, M.、Hino, N.、Yamanaka, T.、Hukushima, H.、Imanishi, T.、uchiyama, Y.、Kodama, T.、Doi, T.：“丙型肝炎病毒核心蛋白与 C