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5 ' transcript replacement in vitro catalyzed by a group I intron-derived ribozyme

机译：5'转录物替代体外由Intron衍生的核酶催化

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摘要

Group I intron-derived ribozymes can perform a variety of catalytic reactions, including the replacement of the 3' end of a mutant RNA transcript with a corrected version of the transcript [Sullenger, B. A., and Cech, T. R. (1994) Nature 371, 619-622]. We now demonstrate in vitro that a ribozyme, derived from a Pneumocystis carinii group I intron, can replace the 5' end of a targeted exogenous RNA with an endogenous RNA. Our model system is a short synthetic mimic of a k-ras transcript, in which substitution mutations at codon 12 are implicated in a host of cancer types. In these experiments, yields of up to 70% were obtained. We analyzed the length dependence of two molecular contacts, P9.0 and P10, that occur between the ribozyme and the exogenous k-ras mimic, and determined that longer, and thus more stable, interactions result in higher product yields. Furthermore, the length of the loop region L1 can substantially influence the yield and the rate of the reaction. These results are a further demonstration that group I intron-derived ribozymes are quite malleable in terms of intermolecular recognition and catalysis, and that these properties can be exploited in developing potentially useful biochemical tools.
机译：I基团衍生的核酶可以进行各种催化反应，包括用矫正形式的转录版[Sullenger，Ba和Cech，TR（1994）自然371,619 -622]。我们现在在体外证明衍生自肺肺炎Carinii族I内含子的核酶可以用内源RNA代替靶向外源RNA的5'末端。我们的模型系统是K-RAS转录物的短合成模拟，其中密码子12处的取代突变涉及一系列癌症类型。在这些实验中，获得高达70％的产率。我们分析了两种分子触点，P9.0和P10的长度依赖性，其在核酶和外源K-Ras之间发生模仿，并确定更长，因此更稳定，相互作用导致更高产物产率。此外，环形区域L1的长度可以基本上影响反应的产率和速率。这些结果是进一步的示范，即在分子间识别和催化方面，I基团Intron衍生的核酶是非常可延展的，并且可以在开发潜在的生化工具中利用这些性质。

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