Komatsu Y, Shirai M, Yamashita S, Ohtsuka E
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Bioorg Med Chem. 1997 Jun;5(6):1063-9. doi: 10.1016/s0968-0896(97)00042-4.
Hairpin ribozymes with high cleavage activities were designed. An extra sequence was introduced at the 3'-end of the hairpin ribozyme to increase the binding to the substrate RNA, as compared to the wild-type hairpin ribozyme. A three-way junction (TWJ) was formed between the newly designed ribozyme and the substrate RNA. The complex with a solid TWJ showed less RNA cleavage activity than the wild-type hairpin ribozyme. However, the ribozyme with a TWJ with five unpaired bases or propandiol phosphate linkers had higher cleavage activity than the parent ribozyme without the TWJ. When a cis-cleavage system, in which the 5'-end of the substrate RNA was conjugated to the 3'-end of the ribozyme, was employed, the complex with the TWJ containing unpaired bases was also cleaved faster than the complex with the solid TWJ. This suggested that these differences in the cleavage activities were derived from the confirmation, and this was proven by nondenaturing gel electrophoresis. The TWJ hairpin ribozyme containing unpaired bases is able to bind strongly with substrate RNAs and to cleave them efficiently. Since the three-way ribozyme presented here is more active than the wild-type ribozyme, this type of ribozyme can serve as a more efficient tool to control RNA activities in vitro and in vivo.
设计了具有高切割活性的发夹状核酶。与野生型发夹状核酶相比，在发夹状核酶的3'端引入了一个额外序列，以增强与底物RNA的结合。新设计的核酶与底物RNA之间形成了一个三向接头（TWJ）。具有稳定TWJ的复合物显示出比野生型发夹状核酶更低的RNA切割活性。然而，具有含五个未配对碱基的TWJ或丙二醇磷酸连接体的核酶比没有TWJ的亲本核酶具有更高的切割活性。当采用一种顺式切割系统，即底物RNA的5'端与核酶的3'端偶联时，含有未配对碱基的TWJ的复合物也比具有稳定TWJ的复合物切割得更快。这表明这些切割活性的差异源自构象，非变性凝胶电泳证明了这一点。含有未配对碱基的TWJ发夹状核酶能够与底物RNA强烈结合并有效切割它们。由于此处呈现的三向核酶比野生型核酶更具活性，这种类型的核酶可作为一种更有效的工具，用于在体外和体内控制RNA活性。
