Tabler M, Homann M, Tzortzakaki S, Sczakiel G
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Crete, Greece.
Nucleic Acids Res. 1994 Sep 25;22(19):3958-65. doi: 10.1093/nar/22.19.3958.
Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.
在催化结构域两侧带有长的靶标特异性反义序列的反式切割锤头状核酶与传统反义RNA具有一些共同特征，因此被称为“催化性反义RNA”。当与靶标RNA复合时，催化结构域5'端的序列形成螺旋I，其3'端的序列形成螺旋III。一种对1型人类免疫缺陷病毒（HIV-1）具有特异性的、超过400个核苷酸的催化性反义RNA，在最初构成128个碱基对螺旋I的臂内被系统地截短。所得核酶分别形成了13、8、5、3、2、1和0个核苷酸的螺旋I，以及大约280个核苷酸的螺旋III。当将它们的体外切割活性与原始催化性反义RNA进行比较时，发现仅三个核苷酸的螺旋I就足以实现完全的内切核酸酶活性。在人类细胞中进行测试时，具有催化活性的构建体抑制HIV-1复制的效果比无活性的构建体高约四倍。当在催化性反义RNA的条件下进行测试时，两侧仅具有8个核苷酸螺旋的传统锤头状核酶在体外无法切割靶标RNA。只有在对核酶底物混合物进行热处理后才能检测到切割活性，这表明短臂锤头状核酶与靶标RNA的结合效率不如催化性反义RNA。仅需三个核苷酸的螺旋I的要求允许采用基于简单PCR的策略来生成不对称锤头状核酶。将讨论不对称设计的优点。
