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Issue 28, 2016
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In vitro repair of a defective EGFP transcript and translation into a functional protein

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Abstract

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.

Graphical abstract: In vitro repair of a defective EGFP transcript and translation into a functional protein

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Supplementary files

Article information


Submitted
13 May 2016
Accepted
09 Jun 2016
First published
09 Jun 2016

Org. Biomol. Chem., 2016,14, 6729-6737
Article type
Paper

In vitro repair of a defective EGFP transcript and translation into a functional protein

D. Balke, A. Becker and S. Müller, Org. Biomol. Chem., 2016, 14, 6729
DOI: 10.1039/C6OB01043A

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