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首页> 外文期刊>Bioorganic and Medicinal Chemistry >Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element
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Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element

机译：针对HIV-1 Rev反应元件的嵌合RNase H功能寡核苷酸

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摘要

Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop ⅡB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop Ⅱ RNA (K_D approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop Ⅱ RNA and have half-lives exceeding 24 h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.
机译：制备了嵌合的寡核苷酸2'-O-甲基核糖核苷酸，该寡核苷酸包含位于中心的连续2'-脱氧核糖核苷酸的贴片并终止于抗核酸酶的3'-甲基膦酸酯核苷酸间键合。该寡核苷酸靶向HIV Rev响应元件（RRE）茎环ⅡBRNA的3'端，该RNA与高亲和力Rev蛋白结合位点相邻，对病毒功能至关重要。热变性实验表明，嵌合寡核苷酸与互补的单链RNA形成了非常稳定的双链体，凝胶电泳迁移率变动分析（EMSA）表明，它们与RRE茎环ⅡRNA（K_D约200 nM）具有高亲和力和特异性结合。 。当在含有10％胎牛血清的细胞培养基中孵育时，该嵌合寡核苷酸可促进RNase H介导的RRE茎环ⅡRNA水解，并具有超过24小时的半衰期。在用p-RRE / CAT和p-Rev哺乳动物表达载体共转染的HEK 293T细胞中，一种嵌合寡核苷酸以300 nM的浓度抑制RRE介导的氯霉素乙酰转移酶（CAT）表达约60％。

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