Medina M F, Joshi S
Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, 150 College Street #212, Toronto, Ontario M5S 3E2, Canada.
Nucleic Acids Res. 1999 Apr 1;27(7):1698-708. doi: 10.1093/nar/27.7.1698.
A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.
一种靶向HIV-1 env编码区的锤头状核酶被表达为人类tRNA3Lys反密码子环的一部分，而不影响tRNA的稳定性或核酶的催化活性。这些tRNA-核酶是从一个文库中分离出来的，该文库设计包含随机序列和可变长度的接头（连接核酶与反密码子环的序列）。在筛选过程中顺式提供核酶靶位点，在后续表征过程中反式提供。具有理想接头组合的tRNA-核酶有望更自由地识别顺式靶位点并进行切割。对切割后的分子进行分离、克隆和表征。鉴定出有活性的tRNA-核酶，并确定了有利于切割的结构特征。所选的tRNA-核酶稳定，切割速率低于或类似于线性锤头状核酶，并且可以由含有RNA聚合酶III的提取物转录。在人CD4+ T细胞系中测试了表达tRNA-核酶的逆转录病毒载体，结果显示其能抑制HIV-1复制。因此，这些基于tRNA3Lys的锤头状核酶在基础研究和应用研究中都应具有重要价值。尤其寻求在HIV-1或HIV-2基因治疗中的应用。
