Design of Nuclease-resistant Antisense Molecules Based on the X-ray Structures of Some Nucleases

基于某些核酸酶X射线结构的抗核酸酶反义分子设计

• 批准号: 11557186
• 负责人: MATSUDA Akira
• 金额: $ 4.22万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557186/
• 关键词: exo-nuclease; endo-nuclease; antisense molecule; radical cyclization; 4'-branched thymidine; oligodeoxyribonucleotide; thermal stability; phosphodiester bond

We have designed nuclease-resistant phosphodiester antisense molecules based on the X-ray crystalographic structures of some restriction enzymes such as Eco R1 and Bam H1. The active sites of these enzymes contain several acidic and basic amino acids residues, which activate water molecules to cleave phosphodiester bonds in DNA.Therefore, if oligonucleotides (ODNs) have certain amino function at the 4'α-position, such ODNs would not be cleaved by such enzymes due to steric and/or charge repulsion. However, to synthesize a branched-sugar nucleoside at the 4'α-position, a new method should be developed. We developed a new radical chemistry with a vinylsilyl or allylsilyl group as a temporary connecting radical acceptor tether to synthesize the required 4'α-branched thymidines. Property of phosphodiester-type ODNs containing 4'α-C-aminomethyl, -ethyl, -propyl, and-N-(2-aminoethyl) carbamoylthymidines (1, 2, 4, and 5, respectively) as a potential antisense molecule are investigated in detail. The nucleosides 1, 2, 4 and 5 were incorporated into octadecamer, [5'-d[MTMTMTMTMTMTMTMTMT]-3', where M is 5-methyl-2'-deoxycytidine], instead of T at various positions. The ODNs containing the modified nucleosides formed more stable duplexes with the complementary DNA than the corresponding unmodified ODN.These ODNs also formed stable duplexes with the complimentary RNA.The ODNs containing the modified nucleosides were significantly resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase (a 3'-exonuclease) and DNase I (an endonuclease) and were also very stable in PBS containing 50% human serum. This is notable, because these consist of natural phosphodiester linkages. Furthermore, the duplexes formed by the ODNs containing the modified nucleosides and their complementary RNAs were good substrates for Escherichia coli RNase H.Thus, these ODNs were identified as a candidate for antisense molecules.

我们已经设计了抗核酸酶的磷酸二酯反义分子的基础上，一些限制性内切酶，如Eco R1和BAM H1的X-射线晶体结构。这些酶的活性位点含有几个酸性和碱性氨基酸残基，它们可以激活水分子切割DNA中的磷酸二酯键，因此，如果寡核苷酸（ODNs）在4 'α-位具有一定的氨基功能，则由于空间和/或电荷排斥作用，这些ODNs不会被这些酶切割。然而，为了在4 'α-位合成支链糖核苷，需要开发新的方法。我们开发了一种新的自由基化学方法，用乙烯基硅烷基或烯丙基硅烷基作为临时连接自由基受体系链来合成所需的4 'α-支链胸苷。详细研究了含4 ′ α-C-氨甲基、-乙基、-丙基和-N-（2-氨乙基）氨甲酰基胸苷（分别为1、2、4和5个）的磷酸二酯型寡核苷酸作为潜在反义分子的性质。将核苷1、2、4和5掺入十八聚体[5 ′-d[MTMTMTMTMTMTMTMT]-3 ′，其中M是5-甲基-2 ′-脱氧胞苷]，而不是在不同位置的T。修饰后的ODNs能与互补DNA形成稳定的双链体，与互补RNA也能形成稳定的双链体，对蛇毒磷酸二酯酶（3 '-核酸外切酶）和DNase I（核酸内切酶）的核酸水解有明显的抵抗力，在含50%人血清的PBS中也很稳定。这是值得注意的，因为它们由天然磷酸二酯键组成。此外，由含有修饰的核苷的ODNs与它们的互补RNA形成的双链体是大肠杆菌RNase H的良好底物，因此，这些ODNs被鉴定为反义分子的候选者。

项目成果

S.Shuto, et al.: "Stereoselective synthesis of α-and β-C-glucosides via radical cyclization with an allylsilyl tether. Control of the stereoselectivity by changing the conformation of the pyranose ring."Tetrahedron Lett.. 41. 4151-4155 (2000)

S.Shuto 等人：“通过烯丙基甲硅烷基系链的自由基环化立体选择性合成 α-和 β-C-葡萄糖苷。通过改变吡喃糖环的构象控制立体选择性。”Tetrahedron Lett.. 41. 4151- 4155 (2000)

I. Sugimoto, et al.: "Synthesis of 4'α-branched thymidines as a new type of antiviral agent"Bioorg. Med. Chem .Lett.. 9. 385-388 (1999)

I. Sugimoto 等人：“作为新型抗病毒剂的 4α-支链胸苷的合成”Bioorg. Med. Chem.Lett.. 9. 385-388 (1999)

S.Shuto, et al.: "Synthesis of 3, 7-anhydro-D-glycero-D-ido-hexitol 1, 5, 6-trisphosphate as a potential IP_3 receptor ligand using a radical cyclization reaction with a vinylsilyl tether as the key step. Conformational restriction strategy using steric r

S.Shuto 等人：“使用乙烯基甲硅烷基系链作为自由基环化反应，合成 3, 7-脱水-D-甘油-D-ido-己糖醇 1, 5, 6-三磷酸作为潜在的 IP_3 受体配体

M.Kanazaki, et al.: "Highly nuclease-resistant phosphodiester-type oligonucleotides containing 4'alpha-C-aminoalkylthymidines form thermally stable duplexes with DNA and RNA. A candidate for potential antisense molecules"J.Am.Chem.Soc.. 122. 2422-2432 (20

M.Kanazaki 等人：“含有 4α-C-氨基烷基胸苷的高度核酸酶抗性磷酸二酯型寡核苷酸与 DNA 和 RNA 形成热稳定双链体。潜在反义分子的候选者”J.Am.Chem.Soc..

Y.Ueno et al.: "Introduction of lipophilic groups into 4'α-C-(2-aminoethyl)thymidine-containing phosphodiester oligodeoxynucleotides and thermal stabilities of the duplexes"Tetrahedron. 56. 7903-7907 (2000)

Y.Ueno 等人：“将亲脂基团引入含磷酸二酯寡脱氧核苷酸和双链体的热稳定性”Tetrahedron 56. 7903-7907 (2000)。