Towards the selection of phosphorothioate aptamers -: Optimizing in vitro selection steps with phosphorothioate nucleotides
Towards the selection of phosphorothioate aptamers -: Optimizing in vitro selection steps with phosphorothioate nucleotides
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DOI：
10.1046/j.1432-1327.2000.01557.x
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发表时间：
2000-08-01
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影响因子：
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通讯作者：
Litvak, S
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文献类型：
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作者：
Andreola, ML;Calmels, C;Litvak, S
给定核酸对蛋白质配体的高亲和力可用于分离病理情况中涉及的酶的特异性抑制剂。后一种性质是SELEX（通过指数富集的配体系统进化）技术的基础。最近，已经使用SELEX方法分离了几种有效的HIV-1复制核酸抑制剂。然而，磷酸二酯寡脱氧核苷酸（PO-ODNs）没有被用作抗病毒剂，因为它们对核酸酶敏感。我们在这项工作中的目标是探索从完全取代的硫代磷酸酯库中选择对HIV-1逆转录酶（RT）和核酸酶抗性都具有强亲和力的寡核苷酸的可能性。HIV-1 TCT从宿主tRNA（Lys）的3'末端启动体内逆转录。尽管硫代磷酸酯ODN（PS-ODN）被认为与蛋白质非特异性结合，但我们以前已经证明，对应于tRNA（Lys）的受体茎的ODN能够特异性抑制HIV-1感染细胞中的HIV-1复制，而在高达10 μ M时不显示细胞毒性。由于SELEX策略需要所选DNA的“体外”转录和逆转录，我们已经通过使用PS-dNTPs和rNTPs分析了可用的PS前体作为模型系统。我们还开发了一种实验程序，以优化在SELEX方法的PCR步骤期间掺入四种PS-dNTPs。在这项工作的过程中，我们已经表明，PS-dGTP是热稳定DNA聚合酶以及HIV-1 RT的强抑制剂。
The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV-1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO-ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV-1 reverse transcriptase (RT) and nuclease resistance. HIV-1 TCT initiates in vivo reverse transcription from the 3' end of a host tRNA(Lys). Although phosphorothioate ODNs (PS-ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNA(Lys) was able to inhibit specifically HIV-1 replication in HIV-1 infected cells, without showing cytotoxicity up to 10 mu M. As the SELEX strategy requires 'in vitro' transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS-dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS-dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS-dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV-1 RT.