TARGETED MRNA CLEAVAGE BY 3 TRNASE AND SMALL GUIDE RNAS

反义和核酶方法显示出抑制作用的巨大希望 不适当的基因表达和最终治疗各种疾病 各州。不幸的是，这些方法有几个缺点 包括将核酸运送到细胞是否存在困难 通过基因治疗或寡核苷酸方法。 含有少至七个核苷酸的寡核苷酸是有效的 在靶向特定的RNA以进行体外切割时，发现了一种3‘RNase 所有的哺乳动物细胞。这种切割需要寡核苷酸 (小引导RNA)在与特定基因精确距离处杂交 二次结构。这项技术已经成功地针对HIV RNA 用于体外卵裂。这项提议的目标是证明mRNAs 在体外和体内都可以被特异性地靶向切割。 该项目的具体目标是：1)证明该方法可以 被用来专门针对功能mRNAs，包括 氯霉素乙酰转移酶(CAT)的体外研究，2)结构图谱 由此产生的CAT mRNA/小引导RNA杂交物研究是否 提议的计算机生成的结构实际上是在体外形成的，以及3) 引入表达短RNA分子的RNA七聚体和/或构建体 它们与报告基因(如CAT)杂交，以确定效率 内源性3‘tRNase在体内特异性降解靶向mRNAs。 与其他基于寡核苷酸的方法相比，这种方法有许多优点 临床上调节不适当基因表达的方法 自只含有7个核苷酸的寡核苷酸以来的情况 与传统的反义方法相比，似乎是有效的 其中含有大于15-20个核苷酸的核苷酸是 必填项。较小的寡核苷酸更容易被 细胞，可能更耐降解，更容易 对其进行化学修饰以增加其稳定性和细胞膜 渗透性。

Antisense and ribozyme methodologies show great promise for inhibiting inappropriate gene expression and ultimately treating various disease states. Unfortunately, these approaches have several drawbacks including difficulty in delivering the nucleic acid to the cell whether by gene therapy or oligonucleotide methods. Oligonucleotides containing as few as seven nucleotides are effective in targeting specific RNAs for cleavage in vitro by a 3' RNase found in all mammalian cells. This cleavage requires that the oligonucleotide (small guide RNA) hybridize at a precise distance from particular secondary structures. This technique has successfully targeted HIV RNAs for cleavage in vitro. The goal of this proposal is to show that mRNAs can be specifically targeted for cleavage in vitro as well as in vivo. The specific aims of the project are to: 1) show that this method can be used to specifically target functional mRNAs including chloramphenicol acetyl transferase (CAT) in vitro, 2) structure map the resulting CAT mRNA/small guide RNA hybrids to investigate whether the proposed computer-generated structures actually form in vitro, and 3) introduce RNA heptamers and/or constructs expressing short RNA molecules which hybridize to reporter mRNAs (e.g CAT), to determine the efficiency of endogenous 3' tRNase to specifically degrade targeted mRNAs in vivo. This approach has many advantages over other oligonucleotide-based methods for modulating inappropriate gene expression in a clinical situation since oligonucleotides containing only seven nucleotides appear to be efficacious in contrast to traditional antisense approaches in which nucleotides containing greater than 15-20 nucleotides are required. Smaller oligonucleotides are much more easily taken up by the cell, may be more resistant to degradation, and are more easily chemically modified to increase their stability and cell membrane permeability.