Lieber A, Strauss M
Max-Planck-Gesellschaft zur Förderung der Wissenschaften, Humboldt University, Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Mol Cell Biol. 1995 Jan;15(1):540-51. doi: 10.1128/MCB.15.1.540.
Inactivation of gene expression by antisense mechanisms in general and by ribozymes in particular is a powerful technique for studying the function of a gene product. We have designed a strategy for expression of ribozymes, for selection of accessible cleavage sites in target RNAs, and for isolation of ribozymes from a library of random sequences flanking the unique sequence of a hammerhead. The expression cassette for ribozyme genes is based on adenovirus-associated RNA. Alternatively, we used polymerase III or the T7 phage transcription machinery. The ribozyme sequences are positioned in the center of a stable stem-loop structure, allowing for a correctly folded ribozyme region within the expressed RNA. A library of ribozyme genes with random sequences of 13 nucleotides on both sides of the hammerhead was generated. As an example, ribozymes which are specific for seven sites within the mRNA or nuclear RNA of human growth hormone were selected and identified. Sequencing of ribozyme genes reamplified from the library confirmed not only the predicted cleavage sites but also the presence of different ribozyme variants in the library. In a test of the ribozyme variants for repression of growth hormone synthesis in a cellular assay, the strongest effect (more than 99% inhibition) was found for the variant with the shortest stretch of complementarity (7 and 8 nucleotides on either side) to the target RNA. This basic strategy seems to be applicable to the selection of suitable target sites and to the isolation of corresponding ribozymes for any mRNA of interest.
一般而言，通过反义机制，特别是通过核酶使基因表达失活，是研究基因产物功能的一项强大技术。我们设计了一种策略，用于核酶的表达、在靶RNA中选择可及的切割位点，以及从锤头状核酶独特序列两侧的随机序列文库中分离核酶。核酶基因的表达盒基于腺相关RNA。另外，我们使用了聚合酶III或T7噬菌体转录机制。核酶序列位于稳定茎环结构的中心，使得在表达的RNA中有一个正确折叠的核酶区域。构建了一个核酶基因文库，其锤头状结构两侧具有13个核苷酸的随机序列。例如，筛选并鉴定了对人生长激素mRNA或核RNA内七个位点具有特异性的核酶。对从文库中重新扩增的核酶基因进行测序，不仅证实了预测的切割位点，还证实了文库中存在不同的核酶变体。在细胞实验中测试核酶变体对生长激素合成的抑制作用时，发现与靶RNA互补性最短（两侧各7和8个核苷酸）的变体具有最强的抑制效果（超过99%）。这一基本策略似乎适用于选择合适的靶位点以及分离针对任何感兴趣mRNA的相应核酶。
