Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding.
Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding.
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DOI：
10.1016/s0021-9258(18)47090-1
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发表时间：
1994-10
期刊：
影响因子：
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通讯作者：
D. Brown;Shin-Heh Kang;S. Gryaznov;L. DeDionisio;O. Heidenreich;S. Sullivan;Xiao Xu;M. Nerenberg
中科院分区：
文献类型：
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作者：
D. Brown;Shin-Heh Kang;S. Gryaznov;L. DeDionisio;O. Heidenreich;S. Sullivan;Xiao Xu;M. Nerenberg
磷酸硫代修饰核苷间键被广泛用于防止血清和细胞中寡脱氧核苷酸（ODN）治疗剂的降解。这种修饰通常会增加ODN的效力，但在许多情况下，它与鲜为人知的非特异性效应的增加有关。在这项研究中，我们发现细胞保留和非特异性蛋白质结合都依赖于寡核苷酸修饰的程度。用荧光素标记的单链（ss）或双链（ds） odn处理的细胞流式细胞术显示，完全磷硫代修饰的odn比3‘端修饰的odn（具有三个3’端磷硫代键）具有更大的细胞相关性。此外，用ss-或ds-探针进行的凝胶移位试验表明，与3'端修饰或未修饰的odn相比，完全磷酸化修饰的odn也表现出更大的细胞质和核蛋白结合。然而，凝胶转移竞争分析显示，与3'端修饰和未修饰的ds- odn相比，完全磷酸化修饰的ds- odn与转录因子的结合完全不具有特异性。这些结果表明，odn的全硫代修饰带来的益处可能会被非特异性蛋白结合和相关序列无关效应的增加所抵消。
Phosphorothioate modification of internucleoside linkages is widely used to prevent degradation of oligodeoxynucleotide (ODN) therapeutic agents in serum and cells. This modification generally increases ODN potency, but in many instances it is associated with an increase of poorly understood nonspecific effects. In this study, we have found that both cellular retention and nonspecific protein binding are dependent upon the extent of the oligonucleotide's modification. Flow cytometry of cells treated with fluorescein-labeled single-stranded (ss) or double-stranded (ds) ODNs demonstrated that fully phosphorothioate-modified ODNs exhibit much greater cellular association than 3'-terminally modified ODNs (with three 3'-terminal phosphorothioate linkages). Additionally, gel shift assays with either ss- or ds-probes showed that fully phosphorothioate-modified ODNs also exhibit much greater cytoplasmic and nuclear protein binding than either 3'-terminally modified or unmodified ODNs. However, gel shift competition assays showed that transcription factor binding by fully phosphorothioate-modified ds-ODNs was completely nonspecific relative to 3'-terminally modified and unmodified ds-ODNs. These results suggest that the benefits derived from full phosphorothioate modification of ODNs may be negated by increases of nonspecific protein binding and associated sequence-independent effects.