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STUDY ON DRUG-DESIGN AND SYNTHESIS OF NOVEL 2-5A-ANTISENSE CHIMERAS

新型2-5A-反义嵌合体的药物设计和合成研究

基本信息

批准号：
12672150
负责人：
KITADE Yukio
金额：
$ 2.11万
依托单位：
GIFU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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/grant/KAKENHI-PROJECT-12672150/
关键词：
2-5A system interferon Rnase L 2-5A-antisense chimera antisense anti-viral drug RNaseL 抗ウイルス作用

项目摘要

Antisense oligonucleotides have been applied extensively for the regulation of cellular and viral gene expression. They hybridize to mRNA targets through Watson-Crick base-pairing and inhibit the translation of mRNA in a sequence-specific manner. On the other hand, a small oligoadenylate containing unique 2',5'-phosphodiester bonds, known as 2-5A, plays a key role in mediating the antiviral effect of interferon. Rnase L, an enzyme found in many eukaryotic cells, is allosterically activated by 2-5A.Few studies were documented to reveal the structure-activity relationship in Rnase L activation by 2-5A derivatives. However, a direct comparison of the potency of these derivatives is not easily made due to the diversity of assay methods. We have now developed a facile method for assaying the activity of Rnase L by the use of non-fusion Rnase L expressed in E. coli and yeast 5S ribosomal RNA as a substrate. These results suggest that modification at the 8-position in the third adenine ring of 2-5A molecule effectively brings about dimerization of Rnase L.We have also reported the synthesis of 8-methyladenosine-containing 2-5 A tetramers and the corresponding antisense chimeras with hydroxyalkyl groups at 5'-phosphates. The phosphates-resistant property and the ability of these oligonucleotides to activate recombinant human Rnase L were studied. The enzyme activated by the 2-5A-antisense chimera, which had the hydroxyethyl group and 8-methyladenosine, cleaved the complementary RNA as efficiently as that activated by the 2-5A-antisense chimera without the hydroxyethyl group and 8-methyladenosine. Thus, the 2-5A-antisense chimera carrying the hydroxyethyl and 8-methyladeosine will be a candidate for novel antisense molecule.

反义寡核苷酸已广泛应用于细胞和病毒基因表达的调控。它们通过Watson-Crick碱基配对与mRNA靶点杂交，并以序列特异性方式抑制mRNA的翻译。另一方面，含有独特的2 '，5'-磷酸二酯键的小寡腺苷酸，称为2-5A，在介导干扰素的抗病毒作用中起关键作用。RNase L是一种存在于许多真核细胞中的酶，它被2- 5A变构激活，但很少有文献报道2-5A衍生物激活RNase L的构效关系。然而，由于测定方法的多样性，不容易对这些衍生物的效价进行直接比较。我们现在已经发展了一种简单的方法来测定RNase L的活性，通过使用在E.大肠杆菌和酵母5S核糖体RNA作为底物。这些结果表明，在2-5A分子的第三个腺嘌呤环的8位上的修饰有效地引起RNase L的二聚化。我们还报道了含8-甲基腺苷的2- 5A四聚体和相应的在5 '-磷酸处具有羟烷基的反义嵌合体的合成。研究了这些寡核苷酸的抗磷酸盐特性和激活重组人RNase L的能力。由具有羟乙基和8-甲基腺苷的2- 5A-反义嵌合体激活的酶与由不具有羟乙基和8-甲基腺苷的2- 5A-反义嵌合体激活的酶一样有效地切割互补RNA。因此，携带羟乙基和8-甲基脱氧核苷的2- 5A-反义嵌合体将成为新型反义分子的候选者。