CELLULAR UPTAKE OF OLIGONUCLEOTIDE ANALOGUES

改善寡核苷酸类似物的细胞摄取代表了一种新的治疗方法。 关于这些生物分子应用于 靶向mRNA或形成三链体结构， 基因组DNA作为控制基因表达的手段。 我们的方法需要对寡核苷酸进行系统性加尾， 在3 '-末端、5'-末端或两者处的单体聚乙二醇 形接头. 古贺博士和朱迪思B。里根最近将各种 聚乙二醇大分子（M.W. 2，000 - 10，000） 可控孔玻璃载体（CPG），并已合成 从这些衍生化的支持物中分离寡核苷酸。 这种做法导致 阿贝替丁-聚乙二醇的简易制备 共轭 所有将聚乙二醇大分子 在锚定至CPG的寡核苷酸的5’末端，失败。 上述 偶联物在聚丙烯酰胺凝胶上具有特征的流动性 电泳，并且不影响与未修饰的 互补寡核苷酸。 修饰的双链体的Tm为 类似于天然DNA双链体。 方便 监测这种缀合物的细胞摄取，Judith B. Regan 已经合成了一种荧光标记， 聚乙二醇和缀合物的磷脂部分。 除了应用于细胞摄取的研究外，PEG- 寡核苷酸缀合物也将与大的（500 bp） 互补的mRNA，以评估在体外，所产生的影响， 聚乙二醇尾部对翻译的空间位阻 这些mRNA相对于适当的控制。

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. Our approach entails the systemic tailing of oligonucleotides with monomeric polyethylene glycols at either the 3'-end, 5'-end or both ends. Dr. Koga and Judith B. Regan have recently coupled various polyethylene glycol macromolecules (M.W. 2,000-10,000) onto a controlled-pore glass support (CPG) and have synthesized oligonucleotides from these derivatized supports. This approach led to the facile preparation of oligonucleotide-polyethylene glycol conjugates. All attempts to couple polyethylene glycol macromolecules at the 5'-end of oligonucleotides anchored to CPG, failed. The above conjugates had characteristic mobility on polyacrylamide gel electrophoresis and have not affected hybridization with unmodified complementary oligonucleotides. The Tms of the modified duplexes were similar to that of the native DNA duplexes. To facilitate the monitoring of the cellular uptake of such conjugates, Judith B. Regan has synthesized a fluorescent marker that will be inserted between the polyethylene glycol and the oligonucleotidic moieties of the conjugates. In addition to be applied to the study of cellular uptake, PEG- oligonucleotide conjugates will also be hybridized to large (500 bp) complementary mRNAs to evaluate in vitro, the effect created by the steric hindrance of the polyethylene glycol tail on the translation of these mRNAs relative to proper controls.