DEVELOPMENT OF ARTIFICIAL RIBONUCLEASE BEARING NOVEL FUNCTIONALIZED ANTISENSE OLIGONUCLEOTIDES

新型功能化反义寡核苷酸的人工核糖核酸酶的开发

基本信息

批准号：
05808051
负责人：
SHINOZUKA Kazuo
金额：
$ 1.15万
依托单位：
GUNMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Novel polyamine derivatives (I-IV) were prepard from tris (2-aminoethyl) amine as a mimic of naturally occurring RNase. These compounds bear imidazole and/or primary amine groups as acid-base catalytic sites along with or without intercalating agent. Among the derivatives, the compound (III) which has a combination of imidazole and primary amine moiety along with the intercalator exhibited most potent RNA cleaving activity. The compound (I) in which an imidazole moiety of (III) was substituted by a primary amine moiety also exhibited remarkably high activity. The compound which lacks the intercalator (II) as well as the compound (IV) which has a longer linker arm for the primary amine compared to (III) were inactive.The mechanism of the RNA cleaving reation by (I) and (III) was investigated by several different experiments. The results suggest that the compounds cleave RNA by hydrolytic mechanism.The active sites of the compound (III), two primary amine groups, wereincorporated in C-5 position of 2'-deoxyuracil. The obtained novel nucleoside was converted to fully protected form of 3'-phosphoramidite and used for the synthesis of oligomer.Two types of oligonucleotides (DNA1 and DNA2) were prepared using the above phosphoramidite. The DNA1 is complementary to 5S rRNA loop region and DNA2 is complementary to the stem region. Both oligomers are 11-merlong and have C-5 modified 2'-deoxyuracil at their 5'-terminus. These oligomers were mixed with 5S rRNA for prolonged period and the results were assayd by denaturing polyacrilamide gel electrophoresis. As the result, DNA1 gave two new fragements which were supposed to be generated by the strand scission of the substrate. On the other hand, DNA2 gave no new bands at all. These results indicate that the hybrid compound consisted of RNA cleaving agent and oligonucleotide will be a useful candidate as a new functionallized antisense agent.

新型的多胺衍生物（I-IV）是从Tris（2-氨基乙基）胺中预启用的，作为天然发生的RNase的模仿。这些化合物带有咪唑和/或原代胺基，作为酸碱催化位点，有或没有插入剂。在衍生物中，具有咪唑和原代胺部分与嵌入剂的组合的化合物（III）表现出最有效的RNA裂解活性。（i）的化合物（i）被原代胺部分取代（III）的咪唑部分也表现出很高的活性。与（iii）相比，缺乏介入剂（II）的化合物（II）以及具有较长级胺的连接臂的化合物（IV）是不活跃的。通过（I）和（iii）的RNA切割裂解的机制是通过多个不同的实验进行了研究的。结果表明，该化合物通过水解机制裂解RNA。化合物（III）的活性位点，两个主要胺基，在2'-脱氧尿素的C-5位置均匀。使用上面的磷氧化岩制备了所获得的新型核苷转化为3'-磷酸氧铁的完全保护形式，用于寡聚体的合成。两种类型的寡核苷酸（DNA1和DNA2）。 DNA1与5s rRNA环区域互补，DNA2与茎区域互补。这两种低聚物均为11分钟，在其5'-末端进行了C-5修饰的2'-脱氧尿嘧啶。这些低聚物与5s rRNA长时间混合，并通过贬低聚ac酰胺凝胶电泳测定结果。结果，DNA1给出了两个新片段，这些片段应该通过底物的链分解产生。另一方面，DNA2根本没有提供新乐队。这些结果表明，杂化化合物由RNA裂解剂和寡核苷酸组成，将是一种有用的候选者，作为一种新功能化的反义剂。