MODULATION OF OPIOID AND NMDA RECEPTOR MRNA BY RIBOZYMES

本拟议补编的具体目标反映了 赠款DA-10337，于1996年6月资助：(1)使用已知的核酶基序 为了在哺乳动物细胞中发挥作用，我们将开发一套靶向核酶 和外部引导序列(EGSS)以实现DOR和 NMDA受体mRNAs。在补充工作中，我们将扩大这一点 人类免疫缺陷病毒基因序列中可被切割的位点的探讨 核酶方法。 (2)在原著和补充工作中，我们将进行测试和比较 这些核酶和EGS元件在试管实验中使用 纯化的成分和亚细胞提取物，以选择 最有希望被引入细胞的候选人。与之相对的是 用细胞受体mRNAs的方法，我们将强调核酶 可合成的针对HIV信使核糖核酸的基序和引导序列 在不使用载体的情况下，通过化学方法传递给细胞。 (3)在最初提出的为期三年的研究方案中，我们计划引入 编码核酶和EGS基序的序列进入细胞环境， 利用逆转录病毒载体系统和培养的神经母细胞瘤细胞系 NG108-15，这些实验仍在轨道上进行。然而，这不太可能 本副刊的研究期限(少于两年)将 允许在HIV抗病毒核酶的情况下实现这一目标 细胞对合成RNA的直接摄取及其向细胞的运输 原子核。正如在最初的研究中一样，我们将利用 过去已经研究出的RNA定量技术 在罗伯逊和因图里西实验室工作了几年。 这些实验的长期目标是达到这样一个点，即 来自人类细胞的核酶成分可以被导入细胞 和动物，以比较它们在实现抗艾滋病毒方面的有效性 效果，通过使用DNA反义技术可以获得的效果 寡核苷酸或载体驱动的核酶从植物中传递 王国。

The specific aims of this proposed supplement reflect the original aims of grant DA-10337, funded in June of 1996: (1) Using ribozyme motifs known to function in mammalian cells, we will develop a set of targeted ribozymes and external guide sequences (EGSs) to achieve cleavage of the DOR and NMDA receptor mRNAs. In the supplementary work, we will expand this approach to sites in HIV mRNA sequences which can be cleaved by the ribozyme approach. (2) In both the original and supplementary work, we will test and compare these ribozyme and EGS elements in test tube experiments, using both purified components and subcellular extracts, in order to selected the most promising candidates for introduction into cells. In contrast to the approach with the cellular receptor mRNAs, we will emphasize ribozyme motifs and guide sequence targeted to HIV mRNA which can be synthesized chemically and delivered to cells without the use of vectors. (3) In original proposal for a three-year study, we planned to introduce sequences encoding ribozyme and EGS motifs into the cellular environment, using a retroviral vector system and the cultured neuroblastoma cell line NG108-15, and these experiments remain on track. However, it is unlikely that the research period of the supplement (less than two years) will allow this aim to be pursued in the case of HIV anti-viral ribozymes for the direct uptake of synthetic RNAs by cells and their transport to the nucleus. As in the original research, we will take advantage of the techniques for RNA quantitation which have been worked out over the past several years in the Robertson and Inturrisi laboratories. The long-term goal of these experiments is to reach the point where these ribozymes elements derived from human cells can be introduced into cells and animals, in order to compare their effectiveness in achieving anti-HIV effects, with those obtainable by techniques using DNA antisense oligonucleotides or vector-drive delivery of ribozymes from the plant kingdom.