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The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several featuresRNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed.

译文

治疗性rna的体内有效性,如反义分子和核酶,依赖于几个特征rna分子需要作为稳定和活性分子在正确的细胞区室中高水平表达。开发 “天然” 小RNA编码基因作为表达盒的机会很高,可以满足这些要求。我们已经研究了利用腺病毒VAI RNA作为表达抗HIV-1核酶的细胞质载体。HIV的保守5' 前导序列被选为靶标,因为它存在于所有病毒转录本中并且高度保守。将锤头状核酶替换为VAI RNA的不同部分,并在非洲爪蟾卵母细胞的体内系统中测试所得嵌合体的积累水平,细胞区隔和在含有La抗原的特定核糖核蛋白中的组装。在体外裂解试验和注射卵母细胞的S100提取物中均发现了不同嵌合体活性的有趣差异,其中可以分析RNP环境中核酶的催化活性。

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