KINETIC CHARACTERIZATION OF INTRAMOLECULAR AND INTERMOLECULAR HAMMERHEAD RNAS WITH STEM-II DELETIONS
KINETIC CHARACTERIZATION OF INTRAMOLECULAR AND INTERMOLECULAR HAMMERHEAD RNAS WITH STEM-II DELETIONS
复制标题
DOI：
10.1073/pnas.91.15.6977
复制
发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
--
作者：
LONG, DM;UHLENBECK, OC
描述了一种获得锤头状核酶在体外转录过程中的分子内切割速率的方法。通过避免RNA纯化和复性，使形成RNA的无活性构象的可能性最小化。通过显示，一个分子内锤头和一个密切相关的分子间锤头在给定的一组条件下以相同的速率裂解，我们证实，这两个反应可能有相同的限速步骤。使用体外选择策略从其中六个随机核苷酸替换茎环II的分子文库中分离活性锤头。取代茎环II的核苷酸的序列和数量对锤头切割活性有很大的影响。从分子内文库中选择的三个序列，当转移到分子间锤头背景中时，它们的相对活性相同。
A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same rate-limiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-loop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.