Study of cellular trafficking and membrane permeability of antisense DNA

本研究合成了针对单纯疱疹病毒Ⅰ型（HSV-1）即刻早期前体mRNA 4/5（immediate early pre-mRNA 4/5，IE pre-mRNA 4/5）的反义DNA，并进行了体内外抗疱疹活性的研究。阐明了反义DNA的序列特异性、膜传递效应以及利用药物传递系统（DDS）增强生物活性。由于反义DNA的序列非特异性活性不可忽略，因此我们对含有连续G序列的反义DNA的抗HIV活性进行了评价，结果发现：1）硫代磷酸寡核苷酸具有最强的抗疱疹活性，其活性与细胞间量有关; 2)反义DNA在感染细胞中的分布与未感染细胞有明显差异。3）以限制性剪接位点为靶点的反义DNA具有较强的抗疱疹病毒活性。4)根据碱基配对的热力学和动力学计算，可以描述反义DNA的序列比活性。5)香叶醇修饰反义DNA的5 '端可增强其抗疱疹病毒活性。牛儿酰胺修饰的反义DNA的均匀细胞分布可能反映了抗疱疹活性。6)阳离子脂质体改变了反义DNA的亚细胞分布。7）序列非特异性生物活性被识别。含有连续G序列的序列显示出较强的抗HIV活性。8)反义DNA对兔疱疹性角膜炎病毒复制有抑制作用。

SummaryWe synthesized antisense DNA toward to immediate early pre-mRNA 4/5 (IE pre-mRNA4/5) of herpes simplex virus type1 (HSV-1) and evaluated anti-herpetic activity both in vitro and in vivo system. We clarified sequence specificity of antisense DNA,membrane delivery effects, and enhancement of biological activity by using drug delivery system (DDS). Since sequence non-specific activity of antisense DNA could not be neglected, anti-HIV activity of antisense DNA containing consecutive G sequence were evaluated.We observed from this study following points ; 1) Phosphorothioate oligonucleotide possessed most potent anti-herpetic activity, which was paralleled with intercellular amount. 2) Cellular distribution of antisense DNA in infected cells were obviously different from that in non-infected cells. From this results, it was suggested that virus infection may enhance the delivery efficiency of antisense DNA.3) We found that antisense DNA targeted limited splicing site revealed potent anti-herpetic activity. 4) Sequence specific activity of antisense DNA can be delineated from calculation based on the thermodynamics and kinetics of base-pairing. 5) 5'end modification with geraniol enhanced anti-herpetic activity of antisense DNA in a sequence specific manncr. Homogeneous cellular distribution of geranil modified antisense DNA may reflect on anti-herpetic activity. 6) Cationic liposomes altered subcellular distribution of antisense DNA.7) Sequence non-specific biological activity was recognized. Sequence containing consecutive G sequence showed potent anti-HIV activity. 8) Inhibition of virus replication by antisense DNA was recognized on rabbit herpes cornea keratitis.