EFFECTS OF DSRNA ADENOSINE DEAMINASE (DRADA) ON ANTISENSE RNA STRATEGY

双链核糖核酸(DsRNA)是在细胞内由几种 对细胞生理学有重要影响的过程。示例 包括信使核糖核酸加工，通过内源基因关闭基因表达 反义RNA和RNA病毒的复制。细胞已经进化成 处理这些分子的新机制，一种dsRNA特异性的 产生碱基对错配，从而解开dsRNA的活动， 通过将许多腺苷残基转化为肌苷。虽然在中国 这种dsRNA腺苷脱氨酶或DRADSA在体内的作用仍然存在 自1987年发现以来一直鲜为人知，它最有可能影响 正义和反义RNA之间形成的RNA双链的命运 干扰DRADA修饰的mRNAs的翻译。 对dsRNA在体内或分子内的命运的精确了解 抑制靶基因翻译的机制是先决条件 应用反义RNA技术开发治疗药物 设计用来阻止病毒感染或治疗肿瘤。这样做的目的是 该项目是为了更好地了解DRADA对反义的影响 RNA策略。 我们最近成功地分离出了DRADA克隆。我们将进行 深入调查DRADA对观念和行为的影响 利用DRADA表达载体构建的反义RNA双链及其命运 纯化的重组DRADA蛋白在我们实验室可用。二 具体目标是： 1.DRADA对dsRNA稳定性的影响 通过导入DRADA条件表达式构建进行研究 转化成反义c-fos RNA表达能 有条件地被强制执行。此外，DRADA对 反义RNA靶向内源性c-fos mRNA的翻译将 被调查。 2.利用纯化的重组DRADA蛋白，我们将在体外检测 DRADA是否会攻击RNA的RNA链：DNA， RNA：硫代修饰的DNA和RNA：O-甲基化-RNA双链。 通过综合所有这些实验的信息，我们的目标是 增加我们对DRADA对dsRNA的影响的理解， 从而，反义RNA策略的成功。

Double-stranded RNA (dsRNA) is produced within cells by several processes that have an important impact on cell physiology. Examples include mRNA processing, switching off gene expression by endogenous antisense RNA, and replication of RNA viruses. Cells have evolved a novel machinery for dealing with these molecules, a dsRNA specific activity that generates base-pair mismatches, and thus unwinds dsRNA, by converting many adenosine residues to inosinne. Although the in vivo role of this dsRNA adenosine deaminase or DRADSA has remained obscure since its discovery in 1987, it is most likely to affect the fate of the RNA duplex formed between sense and antisense RNAs and to interfere with the translation of the mRNAs modified by DRADA. Precise knowledge of the fate of dsRNA in vivo or the molecular mechanism for suppression of targeted genes translation is prerequisite for applicaiton of antisense RNA technology to develop therapeutics designed to block viral infection or to treat tumors. The goal of this project is to better understand the effect of DRADA on the antisense RNA strategy. We have recently succeeded in isolating DRADA clones. We will conduct in-depth investigation on the effect of the DRADA on the sense and antisense RNA duplex and its fate using DRADA expression constructs and purified recombinant DRADA proteins available in our laboratory. Two specific aims are sought: 1. The effect of DRADA on the stability of the dsRNA will be investigated by transfecting a DRADA conditional expression construct into a cell line in which the expression of the antisense c-fos RNA can be manupulated conditionally. In addition, the effect of DRADA on the translation of the antisense RNA targeted endogenous c-fos mRNA will be investigated. 2. Using purifed recombinant DRADA proteins, we will examine in vitro whether DRADA will attack the RNA strand of RNA:DNA, RNA:phosphorothioate modified DNA, and RNA: O-methylated-RNA duplexes. By combining the informaiton from all of these experiments, we aim to increase our understanding of the effect of DRADA on dsRNA and, thereby, the success of the antisense RNA strategy.