Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner.
Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner.
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DOI：
10.1016/s0000-000x(00)00000-0
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
W. Pan;P. Xin;V. Bui;G. Clawson
中科院分区：
文献类型：
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作者：
W. Pan;P. Xin;V. Bui;G. Clawson
核酶有效性的主要限制是靶RNA中可接近位点的定义。尽管已经开发了库选择程序，但它们通常很难执行，并且尚未被广泛采用。在这里，我们描述了一种选择技术，利用一个随机的，积极的锤头状核酶（RZ）库在一个迭代的方式。在非活性条件下进行两轮结合后，将选择的活性Rz文库与靶RNA一起孵育，并在测序凝胶上鉴定切割位点。我们使用人乳头瘤病毒16型E6/E7 mRNA作为靶标进行了该文库选择方案，并构建了靶向所鉴定位点的Rz。Rz靶向的网站确定与此程序通常是高度活跃的体外，更重要的是，他们是高度活跃的细胞培养，而他们的催化失活的同行没有。该方案可用于在相对较短的时间内识别一组潜在的靶位点。
A major limitation to the effectiveness of ribozymes is definition of accessible sites in targeted RNAs. Although library selection procedures have been developed, they are generally difficult to perform and have not been widely employed. Here we describe a selection technology that utilizes a randomized, active hammerhead ribozyme (Rz) library in an iterative manner. After two rounds of binding under inactive conditions, the selected, active Rz library is incubated with target RNA, and the sites of cleavage are identified on sequencing gels. We performed this library-selection protocol using human papillomavirus type 16 E6/E7 mRNA as target and constructed Rz targeted to the identified sites. Rz targeted to sites identified with this procedure were generally highly activein vitroand, more importantly, they were highly active in cell culture, whereas their catalytically inactive counterparts were not. This protocol can be used to identify a set of potential target sites within a relatively short time.