Premium
In vivo selection of mRNA repair ribozymes
Author(s) -
Olson Karen,
Muller Ulrich
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.653.3
Subject(s) - ribozyme , ligase ribozyme , mammalian cpeb3 ribozyme , rna splicing , biology , intron , vs ribozyme , messenger rna , hairpin ribozyme , rna , computational biology , context (archaeology) , genetics , microbiology and biotechnology , gene , paleontology
Group I introns are catalytic RNAs (ribozymes) that can excise themselves from pre‐mRNAs. These ribozymes can be taken out of their pre‐mRNA context and modified to replace the 3’‐terminus of a substrate mRNA with their own 3’‐terminus. To employ these trans ‐splicing ribozymes for therapeutic applications, it is necessary to improve the in vivo mRNA repair efficiency. We developed an in vivo selection method that is able to increase the mRNA repair efficiency of the trans ‐splicing Tetrahymena thermophila ribozyme by selecting from pools with several million ribozyme variants. These ribozyme variants contained 10 or 20 randomized nucleotides in their external guide sequences (EGSs), a region of the ribozyme known to increase splicing efficiency and fidelity. Ribozymes with beneficial EGSs were able to repair a mutated mRNA encoding the chloramphenicol acetyl transferase, and allow growth of the E. coli on medium containing chloramphenicol. Our selections isolated several efficient mRNA repair ribozymes. The predicted secondary structures of the selected EGSs contain several distinct structural motifs. Three of these motifs were previously known, and two motifs are new. Further in vivo selections could allow the generation of ribozymes that repair mRNA efficiently enough for therapeutic applications.