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

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.

Towards Higher Complexity in the RNA World: Hairpin Ribozyme Supported RNA Recombination

“…The evolutionary generation of such aptazymes would be indispensable for a more progressed RNA world, where a more advanced RNA organism needed to regulate its metabolism. This study also once more demonstrates the versatility of the hairpin ribozyme, which owing to its unique cleavage/ligation characteristics is a superior tool for rational design of RNA supported RNA processing reactions for modelling RNA world scenarios, [22,23] and with potential application in molecular biology and medicine [24–26] …”

Engineering of hairpin ribozyme variants for RNA recombination and splicing

“…The reaction is pushed into the desired direction by destabilized binding of the sequence patch to be cut out versus stable binding of the new fragment to be ligated in. This has been achieved in a number of scenarios by bulged‐out nucleotides in the twin ribozyme–substrate complex versus a contiguous helix in the twin ribozyme–product complex, or, alternatively, by mismatches in the twin ribozyme–substrate complex versus a fully base‐paired helix in the twin ribozyme–product complex . A severe drawback of the system is that the twin ribozyme does not show multiple turnover, due to product inhibition.…”