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

反义寡核苷酸已被广泛应用于细胞和病毒基因表达的调控。它们通过Watson-Crick碱基配对与信使核糖核酸目标杂交，并以序列特异性的方式抑制信使核糖核酸的翻译。另一方面，含有独特的2‘，5’-磷酸二酯键的小分子寡腺苷，即2-5A，在干扰素的抗病毒作用中起着关键作用。核糖核酸酶L是一种存在于许多真核细胞中的酶，它被2-5A变构激活。一些研究揭示了2-5A衍生物激活核糖核酸酶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力进行直接比较。我们利用在大肠杆菌中表达的非融合核糖核酸酶L，以酵母5S核糖体核糖核酸为底物，建立了一种测定核糖核酸酶L活性的简便方法。这些结果表明，在2-5A分子的第三个腺嘌呤环上的8位修饰有效地促进了核糖核酸酶L的二聚化。我们还报道了含有8-甲基腺苷的2-5A四聚体和相应的5‘-磷酸羟基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核糖核酸酶L的能力。含有羟乙基和8-甲基腺苷的2-5A-反义嵌合体激活的酶与不含羟乙基和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-甲基鸟苷的2-5A-反义嵌合体将成为新型反义分子的候选分子。

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.