Isolation of active ribozymes from an RNA pool of random sequences using an anchored substrate RNA.

To analyze the structure-function relationship of a ribozyme, RNA molecules with cleavage activities were isolated from a pool of RNAs that have a region of 14 random nucleotides for catalysis by a novel in vitro selection method. Active ribozymes from the pool were selected using a substrate RNA anchored to an agarose bead. After 7 or 8 selection cycles, the pool of the selected molecules showed cleavage activity against the substrate RNA. Structures and catalytic activities of the individual selected RNA molecules were then analyzed by cDNA cloning, in vitro transcription and cleavage reactions. All the active ribozymes were found to catalyze the same sequence-specific cleavage reaction as does the hammerhead ribozyme and contain the conserved nucleotide sequences of the hammerhead ribozymes.