In vitro repair of a defective EGFP transcript and translation into a functional protein.
Twin ribozymes mediate the exchange of a short patch of RNA against an exogenous oligonucleotide within a suitable RNA substrate. Thus, twin ribozymes are promising tools for RNA repair, i.e. for the treatment of genetic disorders at the mRNA level. A number of twin ribozyme-mediated RNA fragment exchange reactions have been successfully demonstrated using short model substrates. Herein we show for the first time a twin ribozyme-mediated in vitro repair of a full-length transcript and translation into a functional protein. The system is based on the repair of a designed mutant EGFP mRNA containing the four-base deletion ΔACTC (190-193). Upon twin ribozyme-mediated replacement of a patch of 15 nucleotides with an externally added repair oligonucleotide (19 mer) the wild type sequence of the EGFP transcript could be restored with 32% yield. This is the first time that such a high twin ribozyme-mediated repair yield, so far observed only for short model substrates, has been obtained for a full-length mRNA. Translation of the repaired EGFP-ΔACTC mRNA produces functional EGFP, as detected by the restored fluorescence.
Ernst-Moritz-Arndt-Universität Greifswald, Institut für Biochemie, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. smueller@uni-greifswald.de.
Darko
D
Balke
Ernst-Moritz-Arndt-Universität Greifswald, Institut für Biochemie, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. smueller@uni-greifswald.de.
Aileen
A
Becker
Ernst-Moritz-Arndt-Universität Greifswald, Institut für Biochemie, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany. smueller@uni-greifswald.de.
Sabine
S
Müller
D001483:Base Sequence; D016368:Frameshift Mutation; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins