Design and specificity of hammerhead ribozymes against calretinin mRNA.
We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.
Ellis
J
J
Department of Physiology, University of Cambridge, UK.
Rogers
J
J
D000818:Animals; D001483:Base Sequence; D064032:Calbindin 2; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA; D005260:Female; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000235:genetics*; D013379:Substrate Specificity
C576511:Calb2 protein, mouse; D064032:Calbindin 2; D016337:RNA, Catalytic; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D004247:DNA