Investigating and improving the specificity of ribozymes directed against the bcr-abl translocation.
Chronic myelogenous leukaemia (CML) is associated with a translocation between the ABL and BCR genes on chromosomes 9 and 22, t(9;22). The resulting transcription and translation products, bcr-abl mRNA and p210bcr-abl, are unique to the malignant cells and as such are ideal targets for specific chemicals or drugs. We have designed hammerhead ribozymes to cleave the two predominant forms of bcr-abl mRNA, b2a2 and b3a2. Synthetic bcr-abl RNA substrates were cleaved by the ribozymes in vitro, but so was a wild-type abl RNA sequence. bcr RNA was not cleaved in vitro and mutant ribozymes showed no cleavage activity. Ribozymes designed to cleave 9 nucleotides (nt) from either of the fusion points were non-specific for the bcr-abl substrate, but a ribozyme designed to cleave 3 nt upstream of the b3a2 fusion point was specific for b3a2 RNA. However, this ribozyme was less efficient than the others. The shortening of one of the ribozymes arms from 10 nt to 4 nt resulted in a ribozyme that was more specific without losing any efficiency. We conclude that it is possible to specifically cleave bcr-abl RNA in vitro by using hammerhead ribozymes.
James
H
H
School of Biological Sciences, University of East Anglia, Norwich, Norfolk, UK.
Mills
K
K
Gibson
I
I
D001483:Base Sequence; D002892:Chromosomes, Human, Pair 22; D002899:Chromosomes, Human, Pair 9; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics*; D013379:Substrate Specificity; D014178:Translocation, Genetic
D016337:RNA, Catalytic; D016044:Fusion Proteins, bcr-abl