Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.
Several synthetic 2'-O-methyl-RNA oligomers and their derivatives have been evaluated for inhibitory effect against HIV-induced cytopathic effect and expression of the virus specific antigen in cultured MT-4 cells. In this study, oligo(2'-O-methyl)ribonucleoside phosphorothioates showed a potent inhibitory activity with size dependency (25-mer showed it at 1 microM), but by contrast both 2'-O-methylribo- and deoxy-oligomers with normal phosphate linkages failed to inhibit. However, it should be noted that the patched oligo(2'-O-methyl)ribonucleotide (20-mer), in which five linkages at 5'- and three linkages at 3'-ends of normal phosphates were replaced with thiophosphates, has recovered the substantial inhibitory effect. These results show that the size of oligomer and phosphorothioate linkages, probably resistant to exolytic nucleases, are essential for exhibiting antiviral activity.
The microbiological synthesis of 9-p-D-arabinofuranosyl adenine (ara-A, an antiviral drug) from adenine and arabinofuranosyluracil (ara-U) is described. Various bacteria, especially Enterobacter aerogenes. Escherichia coli, Erwinia herbicola and Aeromonas salmonicida, were found to be able to transfer the arabinofuranosyl moiety of ara-U to adenine (transarabinosylation) in the presence of inorganic phosphate. The optimum conditions for the transarabinosylation were pH 7.0 and 60°C. No reaction was observed in the absence of inorganic phosphate and its optimum concentration was around 30 mM. Six grams of ara-A was produced in liter of reaction mixture in the presence of wet cell paste of Enterobacter aerogenes AJ 11125. Ara-A formed was precipitated in the reaction mixture and isolated with an 87% yield. Physicochemical data for the compound agreed with those of authentic ara-A.
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