Design of highly specific cytotoxins by using trans-splicing ribozymes
Design of highly specific cytotoxins by using trans-splicing ribozymes
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DOI：
10.1073/pnas.96.7.3507
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发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Haseloff, J
中科院分区：
文献类型：
--
作者：
Ayre, BG;Köhler, U;Haseloff, J
我们设计了基于自剪接I组内含子的核酶，它可以将外显子序列反式剪接到选定的RNA靶标上，从而产生功能性嵌合mRNA，并为基因表达提供高度特异性的触发。我们设计了一种针对广泛存在的植物病原体黄瓜花叶病毒外壳蛋白mRNA的核酶，该核酶将白喉毒素a链的编码序列与目标序列的病毒起始密码子进行反接。白喉毒素A链催化延伸因子2的ADP核糖基化，可导致蛋白质翻译停止。在酿酒酵母模型系统中，核酶的表达可以特异性抑制表达病毒mRNA的细胞的生长。靶剪接位点的点突变减轻了这种核酶介导的毒性。增加核酶与靶标之间碱基配对的程度可以显著增加细胞毒素的特异性表达，降低体内毒性。反式剪接核酶可能为工程病毒抗性和治疗细胞毒素提供一类新的试剂。
We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus, The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity invivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.