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

双链RNA（dsRNA）是由几种RNA酶在细胞内产生的。 对细胞生理学有重要影响的过程。 示例 包括mRNA加工、通过内源性转录因子关闭基因表达 反义RNA和RNA病毒的复制。 细胞已经进化出 处理这些分子的新机制，一种dsRNA特异性的 产生碱基对错配的活性，从而解旋dsRNA， 通过将许多腺苷残基转化为肌苷。 虽然在 这种dsRNA腺苷脱氨酶或DRADSA的体内作用仍然存在 自1987年发现以来，它一直默默无闻，最有可能影响到 正义和反义RNA之间形成的RNA双链体的命运， 干扰由DRDA修饰的mRNA的翻译。 精确了解dsRNA在体内的命运或分子水平， 抑制靶基因翻译的机制是先决条件 应用反义RNA技术开发治疗方法 用来阻止病毒感染或治疗肿瘤。 这个目标 项目是为了更好地了解DRDA对反义核酸的影响， RNA策略 我们最近成功地分离出了DRDA的克隆体。 我们会进行 深入调查DRDA对感觉和 反义RNA双链体及其命运， 纯化的重组DRDA蛋白。 两 具体目标如下： 1. DRDA对dsRNA稳定性的影响将是 通过构造DRDA条件表达式来研究 导入反义c-fos RNA表达的细胞系， 有条件地操纵。 此外，裁军事务部对 以内源性c-fos mRNA为靶点的反义RNA的翻译， 追究 2.使用纯化的重组DRDA蛋白，我们将在体外检测 DRDA是否会攻击RNA：DNA的RNA链， RNA：硫代磷酸酯修饰的DNA，和RNA：O-甲基化-RNA双链体。 通过结合所有这些实验的信息，我们的目标是 增加我们对DRDA对dsRNA的影响的理解， 因此，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.