CELLULAR UPTAKE OF OLIGONUCLEOTIDE ANALOGUES

改善细胞对寡核苷酸类似物的摄取代表着一种 关于这些生物分子在生物医学中应用的关键问题 靶向mRNAs或与基因组形成三链结构 DNA作为一种控制基因表达的手段。与改进相关的研究 细胞对寡核苷酸类似物的摄取一直很少，而且很差。 只要涉及到参数的细化，就执行。 我们的方法包括系统地拖尾寡核苷 已知的抗HIV生物活性的硫代磷酸盐，含单体 3‘端、5’端或两端的聚乙二醇及其含量测定 对于慢性感染H9中的无尾硫代低聚物 细胞。 到目前为止，我们已经合成了四种不同的寡核苷硫代硫酸酯 与HIV rev基因编码的mRNA互补 在两端共价结合到1，2，3或4己二醇 单体(S)这些寡核苷酸已被提纯到均一状态。 可变性的聚丙烯酰胺凝胶，可用于生物评价。 所有这些寡核苷酸类似物都形成了稳定的DNA双链 它们相对于无尾寡核苷的互补DNA序列 硫代磷酸盐。(双链在260 nm处具有相同的TM)。 然而，需要注意的是，六甘醇单体是 通过磷酸二酯桥连接在一起。要正确研究 这些生物分子的行为增加了电荷，寡核苷 硫代磷也将在3‘-端进行衍生，不带电荷 不同大小的聚乙二醇分子(分子量1,000-8,000)。朱迪思 B.里根已经提纯了一种硫代低聚物，它与一种 相对较大的聚乙二醇单体(相对分子质量2,500)。TM实验 目前正在进行中，NCI的Mitsuya博士将执行所有 与这些新型生物分子相关的生物检测。

Improving the cellular uptake of oligonucleotide analogues represents a critical issue with respect to the application of these biomolecules in the targeting of mRNAs or in the formation of triplex structures with genomic DNA as a means to control gene expression. Studies related to improve cellular uptake of oligonucleotide analogues have been scarce and poorly executed as far as refinement of the parameters is concerned. Our approach involves the systematic tailing of an oligonucleoside phosphorothioate of known biological activity against HIV, with monomeric polyethylene glycols at either the 3'-end, 5'-end or both ends and assay for the untailed phosphorothioate oligomer in chronically infected H9 cells. So far we have synthesized four different oligonucleoside phosphorothioates complementary to the mRNA encoded by the rev gene of HIV each of them covalently bound at both ends to 1, 2, 3, or 4 hexaethylene glycol monomer(s). These oligonucleotides have been purified to homogeneity on denaturing polyacrylamide gels and are ready for biological evaluation. All of these oligonucleotide analogues formed stable DNA duplexes with their complementary DNA sequences relative to the untailed oligonucleoside phosphorothioates. (The duplexes have the same Tm at 260 nm). It is to be noted, however, that the hexaethylene glycol monomers were linked together by phosphodiester bridges. To properly study the effect of added charges on the behavior of these biomolecules, oligonucleoside phosphorothioates will also be derivatized at the 3'-end with uncharged polyethylene glycol molecules of varying sizes (M.W. 1,000-8,000). Judith b. Regan has already purified a phosphorothioate oligomer conjugated to a relatively large polyethylene glycol monomer (M.W. 2,500). Tm experiments are currently being conducted., Dr. Mitsuya of the NCI will perform all the biological assays pertaining to these novel biomolecules.