The virtues of self-binding: high sequence specificity for RNA cleavage by self-processed hammerhead ribozymes.
Naturally occurring hammerhead ribozymes are produced by rolling circle replication followed by self-cleavage. This results in monomer-length catalytic RNAs which have self-complementary sequences that can occupy their trans -binding domains and potentially block their ability to cleave other RNA strands. Here we show, using small self-processed ribozymes, that this self-binding does not necessarily inhibit trans -cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt circular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Rolling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of complementarity or bind itself with 4+5 nt of self-complementarity by folding back its ends to form hairpins. Kinetics of trans -cleavage of short complementary and mismatched RNAs were measured under multiple turnover conditions, in comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-complementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k (cat)/ K (m)almost the same as MDR40, but with discrimination against mismatches up to 20 times greater. Based on folding predictions, a second self-processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for increasing sequence specificity of ribozymes.
Ohmichi
T
T
Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
Kool
E T
ET
D020137:Base Pair Mismatch / Q000235:genetics; D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D004270:DNA, Circular / Q000235:genetics; D019450:Genes, MDR / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D017354:Point Mutation / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000235:genetics
D004270:DNA, Circular; D016337:RNA, Catalytic; D012313:RNA