Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA

The trans insertion-splicing reaction, catalyzed by a group 1 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 OF 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 intorn-derived ribozyme being capable of catalyzing the insertion of a modified Oligonucleotide into RNA. (c) 2008 Elsevier Inc. All rights reserved.