STUDY OF BINDING ABILITY OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES WITH PROTEIN

我们已经认识到，当目标位点被限制在剪接位点附近时，硫代寡核苷酸（S-ODN）显示出有效的抗疱疹活性。这表明S-ODN干扰了剪接所必需的保守结构，从而显示出强大的抗疱疹活性。这解释了为什么S-ODN靶向剪接位点显示出有效的抗疱疹活性。然而，有报道称富含G的序列显示出序列的非特异性生物活性。S-ODN可能揭示了另一种基于强蛋白结合活性的机制。S-ODN与血清蛋白的结合率为86.6%，与磷酸二酯寡核苷酸（D-ODN）的结合率为21.6%。直接与病毒结合的活性，S-ODN大于50%，D-ODN小于5%。13 mer S-ODN在感染早期对病毒进入细胞有抑制作用，而D-ODN没有。S-ODN的CD谱暗示g -四重奏结构，但其生物活性与高维结构之间的关系尚不清楚。此外，我们还尝试利用阳离子脂质体增强S-ODN的抗疱疹活性。阳离子脂质体是增强D-ODN抗疱疹活性的有效工具，而对SOD则没有作用。其原因之一可能是S-ODN具有较强的蛋白结合活性。从本研究中可以看出，当S-ODN含有富含g的序列时，可能涉及反义方式以外的其他机制。为了提高S-ODN的生物活性，应谨慎选择给药系统。

We've recognized that phosphorothioate oligonucleotides (S-ODN) showed potent anti-herpetic activities when target site was limited near the splicing site. It was implied that S-ODN interfered the conservative structure which could be essential for the splicing, thus showed potent anti-herpetic activities. This was one explanation why S-ODN targeted splicing site showed potent anti-herpetic activities. However, it has been reported that G rich sequences reveal sequence non-specific biological activities. S-ODN may reveal another mechanism which might be based on strong protein binding activities. S-ODN strongly bound to serum protein at the ratio of 86.6%, while 21.6% for phosphodiester oligonucleotides (D-ODN). Binding activities which directly bound to the virus was more than 50% for S-ODN, on the other hand it was less than 5% for D-ODN.Thirteen mer S-ODN showed inhibitory effect on virus entry into the cell at the early stage of infection, while D-ODN did not. CD spectrum of S-ODN implied G-quartet structure, however, the relation between biological activities and higher dimension structure could not be clear.Moreover, we tried to enhance the anti-herpetic activities of S-ODN by using cationic liposomes. Cationic liposomes is useful tool to enhance the anti-herpetic activities of D-ODN, while it was not the case for SOD.One of the reason might be strong protein binding activities of S-ODN.From this study, the caution is proposed when S-ODN containing G-rich sequences might involve another mechanism other than antisense manner. We should carefully select the drug delivery system to enhance the biological activities of S-ODN.