Brown D A, Kang S H, Gryaznov S M, DeDionisio L, Heidenreich O, Sullivan S, Xu X, Nerenberg M I
Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1994 Oct 28;269(43):26801-5.
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.
核苷间连接的硫代磷酸酯修饰被广泛用于防止寡脱氧核苷酸（ODN）治疗剂在血清和细胞中降解。这种修饰通常会提高ODN的效力，但在许多情况下，它与一些难以理解的非特异性效应的增加有关。在本研究中，我们发现细胞保留和非特异性蛋白质结合均取决于寡核苷酸的修饰程度。用荧光素标记的单链（ss）或双链（ds）ODN处理细胞的流式细胞术表明，完全硫代磷酸酯修饰的ODN比3'-末端修饰的ODN（具有三个3'-末端硫代磷酸酯连接）表现出更强的细胞结合能力。此外，使用ss-或ds-探针的凝胶迁移试验表明，完全硫代磷酸酯修饰的ODN比3'-末端修饰或未修饰的ODN也表现出更强的细胞质和核蛋白结合能力。然而，凝胶迁移竞争试验表明，相对于3'-末端修饰和未修饰的ds-ODN，完全硫代磷酸酯修饰的ds-ODN与转录因子的结合完全是非特异性的。这些结果表明，ODN完全硫代磷酸酯修饰所带来的益处可能会因非特异性蛋白质结合的增加及相关的序列非依赖性效应而被抵消。
