A New Type of Hairpin Ribozyme Consisting of Three Domains

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Abstract
We have constructed a new hairpin ribozyme with three stem−loop domains. In the ribozyme, another domain (domain I‘) was connected to the 3‘-end of domain II of the parent hairpin ribozyme, and the new ribozyme can be trimmed after transcription from the DNA template using T7 RNA polymerase. Since a mutant ribozyme containing a substitution at the essential base in domain I‘ lacked the 3‘-trimming reaction, the autoprocessing activity was proved to be derived from the catalytic reaction, similar to the wild-type ribozyme. Furthermore, the structure of the cleavage site from the self-trimming reaction was identified as a 2‘,3‘-cyclic phosphate, which is the same as that of the wild-type. The processed ribozyme was designed to cleave an external substrate RNA derived from the mRNA of the human inducible nitric oxide synthase and was proved to cleave at the expected, unique site. The hairpin ribozyme containing the three-domains exhibited the 3‘-self-trimming activity even in a runoff transcription reaction from the plasmid harboring the ribozyme gene with the three domains. The new type of hairpin ribozyme thus obtained has three stem−loop domains and is able to act as a catalytic RNA for both cis and trans cleavage. These ribozymes are of interest from the point of the structure−function relationship of the hairpin ribozyme and provide an important insight into over understanding of the role of the domain−domain interaction in the catalytic activity.

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