Modification and application of self-trimming hairpin ribozymes to targeting a transcribed RNA in vitro

The three-domain hairpin ribozyme was improved by increasing the number of linker bases connecting domain I' with domain II from six to seven, and a new ribozyme was designed to release a trans-acting ribozyme that is able to bind with target RNAs by three hybridizing arms. The trans-cleavage activities of the trimmed ribozyme were used to cleave a long target RNA, with 85 bases transcribed from a synthetic gene encoding induced nitric oxide synthase (iNOS). Although the ribozyme with three-hybridizing arms did not efficiently cleave the long target RNA, the overall activity, from self-trimming to the trans-cleavage reaction of the target RNA, was higher than that of the ribozyme with two hybridizing arms.