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

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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.