Selection in vitro of novel ribozymes from a partially randomized U2 and U6 snRNA library.
Combinatorial libraries related to spliceosomal U2 and U6 snRNAs were tested for catalytic reactions typical of the splicing of nuclear pre-mRNAs. Ribozymes with four different activities were selected based on covalent bond formation to a substrate RNA. The first activity was reversible self-cleavage; ribozymes self-cleaved then ligated the 5'-hydroxyl group of the substrate oligonucleotide to their 2',3'-cyclic phosphate intermediate. The second activity was 2',5'-branch formation by the attack of a substrate 2'-hydroxyl group on the 5'-terminal triphosphate of the ribozyme transcript, releasing pyrophosphate. The third ribozyme activity was similar to reversible self-cleavage but was a three-step reaction. This ribozyme self-cleaved, then cleaved the substrate in trans, and then ligated the substrate 3' cleavage product to its cyclic phosphate intermediate. This three-step pathway shares similarities with the pathway of tRNA splicing. The fourth activity was 2',3'-branch formation; to form this unusual branch, a 2'-hydroxyl of the substrate attacked an internal phosphate of the ribozyme, releasing an oligonucleotide leaving group. The isolation of branching activities by the in vitro selection protocol was unanticipated and was due to surprising properties of reverse transcriptase, which can read through 2',5'- or 2',3'-branches and efficiently perform non-templated intramolecular jumps.
Tuschl
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Center for Cancer Research and 1,2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Sharp
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Bartel
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D001483:Base Sequence; D001665:Binding Sites; D002678:Chimera; D005091:Exons; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D012342:RNA, Small Nuclear