Prislei S, Buonomo S B, Michienzi A, Bozzoni I
Istituto Pasteur, Fondazione Cenci-Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, Roma, Italy.
RNA. 1997 Jun;3(6):677-87.
The in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA 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.
治疗性RNA（如反义分子和核酶）的体内有效性依赖于几个特征：RNA分子需要在正确的细胞区室中高水平表达，成为稳定且有活性的分子。利用“天然”小RNA编码基因作为表达盒很有可能满足这些要求。我们研究了腺病毒VAI RNA作为细胞质载体来表达针对HIV-1的核酶的用途。选择HIV保守的5'前导序列作为靶点，因为它存在于所有病毒转录本中且高度保守。将锤头状核酶替换到VAI RNA的不同部分，然后在非洲爪蟾卵母细胞的体内系统中测试所得嵌合体在特定含La抗原的核糖核蛋白颗粒中的积累水平、细胞区室化和组装情况。在体外切割试验以及注射卵母细胞的S100提取物中均发现不同嵌合体的活性存在有趣差异，在S100提取物中可以分析核酶在核糖核蛋白环境中的催化活性。
