MODULATION OF OPIOID AND NMDA RECEPTOR MRNA BY RIBOZYMES

这一拟议补编的具体目标反映了 DA-10337基金，1996年6月资助：（1）使用已知的核酶基序， 在哺乳动物细胞中的功能，我们将开发一套靶向核酶 和外部指导序列（EGS）以实现DOR的切割， NMDA受体mRNA。在补充工作中，我们将扩大这一点， HIV mRNA序列中可被切割的位点的方法 核酶方法。 (2)在原有的和补充的工作中，我们将测试和比较 这些核酶和EGS元件在试管实验中， 纯化的组分和亚细胞提取物，以选择 最有前途的候选人引入细胞。相对于 与细胞受体mRNA的方法，我们将强调核酶 可以合成的靶向HIV mRNA的基序和指导序列 通过化学方式并在不使用载体的情况下递送至细胞。 (3)在最初的三年研究建议中，我们计划引入 编码核酶和EGS基序的序列进入细胞环境， 使用逆转录病毒载体系统和培养的神经母细胞瘤细胞系 NG 108 -15，这些实验仍然在轨道上。然而， 补充材料的研究期（不超过两年）将 允许在HIV抗病毒核酶的情况下追求这一目标， 细胞直接摄取合成RNA并将其转运至 原子核在最初的研究中，我们将利用 过去已经研究出的RNA定量技术 在Robertson和Inturrisi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， 可以将来自人细胞的核酶元件引入细胞中 和动物，以比较它们在实现抗艾滋病毒方面的有效性， 效果，与那些可通过使用DNA反义技术 寡核苷酸或载体驱动的核酶从植物的递送 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.