Hammann C, Tabler M
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion/Crete, GR-71110, Greece.
Methods. 1999 Jul;18(3):273-80. doi: 10.1006/meth.1999.0784.
Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5' end the catalytic domain and at its 3' end a relatively long antisense flank that is complementary to the target RNA. Asymmetric hammerhead ribozymes can be constructed via polymerase chain reaction amplification, and rules are provided on how to select the DNA oligonucleotides required for this reaction. In addition to details on construction, we describe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to minimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determination of the cleavage rate constant. Testing for efficient cleavage is also a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for target suppression in living cells.
大多数想要抑制特定基因的研究人员主要关注核酶技术的应用，而非其作用机制的细节。本文提供了一些背景信息，并描述了一种生成和测试特殊设计核酶（即不对称锤头状核酶）的直接策略。这种锤头状核酶在其5'端携带催化结构域，在其3'端携带与靶RNA互补的相对较长的反义侧翼。不对称锤头状核酶可通过聚合酶链反应扩增构建，并给出了如何选择该反应所需DNA寡核苷酸的规则。除了构建细节，我们还描述了如何在体外测试不对称锤头状核酶与靶RNA的结合，以便可以选择和优化RNA构建体，使其能与靶RNA快速杂交。该测试可使人们将由靶RNA二级结构引起的结合问题降至最低。此外，我们还描述了体外切割试验和切割速率常数的测定。测试有效切割也是该技术可靠且成功应用的先决条件。精心挑选的RNA最终用于活细胞中的靶标抑制时会更有前景。
