In vitro selection of active ribozyme sequences of hammerhead type

Abstract

The hammerhead ribozyme consists of three duplex stems and three internal loops. The internal loop sequences between the stems are conserved: a core of two non-helical segments (7 bases between stems I and II; 3 bases between stems II and III) and an unpaired base at the cleavage site (between stems III and I). Although previous results with single and double mutants suggested that none of the core bases could be substituted without losing activity, its recently determined three-dimensional structure led us to try to isolate hammerhead ribozyme sequences that are quite different from the conserved one. Our approach was to separate the active sequences from the inactive ones in a large pool of random sequence RNAs and to amplify the active ones to characterize. DNA were randomly synthesized and a poly(A) tail was added to their 3`` termini. The biotinylated oligo(dT) was used to remove polyadenylated RNAs that contained inactive sequences and 3`` cleavage fragments of active ribozymes. The 5`` cleavage fragments containing active sequences were supposed to remain and amplified using RT-PCR. Then, the resulting DNAs were subjected to several cycles of transcription by phage T7 RNA polymerase, selection by biotinylated oligo(dT) and amplification by RT-PCR. As a result, a wild type sequence and a mutant sequence that hold catalytic activity, although not greater than that of the wild type, were isolated.