Taylor N R, Kaplan B E, Swiderski P, Li H, Rossi J J
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Nucleic Acids Res. 1992 Sep 11;20(17):4559-65. doi: 10.1093/nar/20.17.4559.
Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.
在发现RNA具有位点特异性切割活性之后，人们对设计和测试作为替代遗传工具和治疗剂的反式作用催化RNA产生了浓厚兴趣。我们一直在开发对HIV-1 RNA具有靶标特异性的催化RNA或核酶，并一直在探索化学合成作为其生产方法之一。为此，我们化学合成并实验分析了锤头型核酶非酶部分为DNA、酶核心为RNA的嵌合催化剂。在体外分析了锤头型核酶不同茎中DNA替代RNA后的动力学效率。本研究中使用的一种嵌合核酶，含有24个能与HIV-1 gag靶标进行碱基配对相互作用的DNA碱基，但催化中心和茎环II中仍保留RNA，其kcat值比全RNA对应物高六倍。这种活性增加似乎是产物解离增强的直接结果。有趣的是，一种茎环II（将催化核心分开）由DNA组成的嵌合核酶，其切割活性显著降低，这表明核酶该区域的DNA会对核酶的催化功能产生负面影响。通过阳离子脂质体转染到人T淋巴细胞中的DNA-RNA嵌合核酶比其全RNA对应物更稳定。在对Km没有显著影响的情况下，催化周转率和稳定性的提高使嵌合核酶成为治疗剂的理想候选物。
