Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA.
The trans insertion-splicing reaction, catalyzed by a group I intron-derived from Pneumocystis carinii, was recently developed for the site-specific insertion of a segment of RNA into a separate RNA substrate. The molecular determinants of this reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. To demonstrate proof-of-concept, we now report that the P. carinii ribozyme can except modified oligonucleotides as substrates for catalyzing the trans insertion-splicing reaction. Oligonucleotides that contain one or more sugar modifications (deoxy or methoxy substitution), a backbone modification (phosphorothioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in this reaction. Apparently, trans insertion-splicing is a unique and viable reaction for the site-specific incorporation of modified oligonucleotides into RNAs. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a modified oligonucleotide into RNA.
Dotson
P Patrick
PP
Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Frommeyer
Kristen N
KN
Testa
Stephen M
SM
D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D005656:Fungal Proteins / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D007438:Introns; D008954:Models, Biological; D008957:Models, Genetic; D009841:Oligonucleotides / Q000737:chemistry*; D045363:Pneumocystis carinii / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry*; D013379:Substrate Specificity; D020040:Trans-Splicing
D005656:Fungal Proteins; D009841:Oligonucleotides; D016337:RNA, Catalytic; D012313:RNA