Ishizaka M, Ohshima Y, Tani T
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Biochem Biophys Res Commun. 1995 Sep 14;214(2):403-9. doi: 10.1006/bbrc.1995.2301.
To analyze the structure-function relationship of a ribozyme, RNA molecules with cleavage activities were isolated from a pool of RNAs that have a region of 14 random nucleotides for catalysis by a novel in vitro selection method. Active ribozymes from the pool were selected using a substrate RNA anchored to an agarose bead. After 7 or 8 selection cycles, the pool of the selected molecules showed cleavage activity against the substrate RNA. Structures and catalytic activities of the individual selected RNA molecules were then analyzed by cDNA cloning, in vitro transcription and cleavage reactions. All the active ribozymes were found to catalyze the same sequence-specific cleavage reaction as does the hammerhead ribozyme and contain the conserved nucleotide sequences of the hammerhead ribozymes.
为了分析核酶的结构-功能关系，通过一种新型的体外筛选方法，从具有14个随机核苷酸区域用于催化的RNA池中分离出具有切割活性的RNA分子。使用锚定在琼脂糖珠上的底物RNA从该池中筛选出活性核酶。经过7或8个筛选循环后，所选分子池对底物RNA表现出切割活性。然后通过cDNA克隆、体外转录和切割反应分析各个所选RNA分子的结构和催化活性。发现所有活性核酶都催化与锤头状核酶相同的序列特异性切割反应，并含有锤头状核酶的保守核苷酸序列。
