CONSTRUCTION OF HAIRPIN RIBOZYMES WITH A 3-WAY JUNCTION

Hairpin ribozymes with high cleavage activities were designed. An extr a sequence was introduced at the 3'-end of the hairpin ribozyme to inc rease the binding to the substrate RNA, as compared to the wild-type h airpin ribozyme. A three-way junction (TWJ) was formed between the new ly designed ribozyme and the substrate RNA. The complex with a solid T WJ showed less RNA cleavage activity than the wild-type hairpin ribozy me. However, the ribozyme with a TWJ with five unpaired bases or propa ndiol phosphate linkers had higher cleavage activity than the parent r ibozyme without the TWJ. When a cis-cleavage system, in which the 5'-e nd of the substrate RNA was conjugated to the 3'-end of the ribozyme, was employed, the complex with the TWJ containing unpaired bases was a lso cleaved faster than the complex with the solid TWJ. This suggested that these differences in the cleavage activities were derived from t he conformation, and this was proven by nondenaturing gel electrophore sis. The TWJ hairpin ribozyme containing unpaired bases is able to bin d strongly with substrate RNAs and to cleave them efficiently. Since t he 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. (C) 1997 Elsevier Scienc e Ltd.