Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase
Combinatorial selection, inhibition, and antiviral activity of DNA thioaptamers targeting the RNase H domain of HIV-1 reverse transcriptase
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DOI：
10.1021/bi0507074
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发表时间：
2005-08-02
期刊：
影响因子：
2.9
通讯作者：
Gorenstein, DG
中科院分区：
文献类型：
--
作者：
Somasunderam, A;Ferguson, MR;Gorenstein, DG
尽管人类免疫缺陷病毒-1逆转录酶（HIV-1 RT）的RNase H在病毒增殖中起关键作用，但仅报道了几种RNase H的抑制剂。使用体外组合选择方法和HIV RT的RNA酶H结构域，我们选择了抑制RNA酶H活性和病毒复制的双链DNA硫代适体（具有选定硫代磷酸骨架取代的适体）。所选择的硫代适体序列具有非常高比例的G残基。所选硫代适体的共有序列显示在序列的5 '端由单个残基分开的G簇。凝胶电泳迁移率变动分析和核磁共振光谱表明，所选择的硫代适体结合到分离的RNA酶H结构域，但没有结合到结构相似的RNA酶H从大肠杆菌。先导硫代适体R12-2显示出与HIV-1 RT的特异性结合，结合常数（K-d）为70 nM。硫代适体抑制完整HIV-1 RT的RNA酶H活性。在细胞培养物中，硫代适体R12-2（0.5 μ g/mL）的转染显著抑制病毒产生，并在R12-2浓度范围为0.03至2.0 μ g/mL（IC 50 < 100 nM）时表现出抑制的剂量反应。在广泛的病毒接种物中也观察到抑制，范围从0.0005至0.05的感染复数（moi），在高moi下病毒产生水平降低超过50%。病毒的抑制作用与AZT相当，
Despite the key role played by the RNase H of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) in viral proliferation, only a few inhibitors of RNase H have been reported. Using in vitro combinatorial selection methods and the RNase H domain of the HIV RT, we have selected double-stranded DNA thioaptamers (aptamers with selected thiophosphate backbone substitutions) that inhibit RNase H activity and viral replication. The selected thioaptamer sequences had a very high proportion of G residues. The consensus sequence for the selected thioaptamers showed G clusters separated by single residues at the 5'-end of the sequence. Gel electrophoresis mobility shift assays and nuclear magnetic resonance spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. The lead thioaptamer, R12-2, showed specific binding to HIV-1 RT with a binding constant (K-d) of 70 nM. The thioaptamer inhibited the RNase H activity of intact HIV-1 RT. In cell culture, transfection of thioaptamer R12-2 (0.5 mu g/mL) markedly inhibited viral production and exhibited a dose response of inhibition with R12-2 concentrations ranging from 0.03 to 2.0 mu g/mL (IC50 < 100 nM). Inhibition was also seen across a wide range of virus inoculum, ranging from a multiplicity of infection (moi) of 0.0005 to 0.05, with a reduction of the level of virus production by more than 50% at high moi. Suppression of virus was comparable to that seen with AZT when moi