Design and analysis of hammerhead ribozyme activity against an artificial gene target.
Design and analysis of hammerhead ribozyme activity against an artificial gene target.
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DOI：
10.1007/978-1-62703-730-3_5
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发表时间：
2014
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影响因子：
--
通讯作者：
Fraser, Malcolm J Jr
中科院分区：
文献类型：
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作者：
Carter, James R;Nawtaisong, Pruksa;Balaraman, Velmurugan;Fraser, Malcolm J Jr
通常进行体外裂解测定，以正确评估锤头核酶 (HHR) 对目标 RNA 分子（如登革热病毒 RNA 基因组）的催化活性。这些实验是为了对无细胞系统中的 HHR 催化进行初步评估，并通过用琼脂糖凝胶电泳代替 SDS-PAGE 进行了简化。替代迁移率测定可以在没有放射性同位素的情况下以更快速的方式分析核酶。在这里，我们描述了 HHR 和相应靶标从 T7 启动质粒到 RNA 分子的体外转录，从而通过体外裂解测定分析 HHR 针对 RNA 靶标的活性。
In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like the dengue virus RNA genomes. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.