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Published on: July 22, 2014
Cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and has strong impact on
Michael Andäng1, Catharina Maijgren-Steffensson, Jorma Hinkula
1Division of Molecular Neurobiology, Karolinska Institutet, MBB, 171 77 Stockholm, Sweden. michael.andang@mbb.ki.se
Abstract:
Trans-cleaving hammerhead or hairpin ribozymes were expressed in transgenic mice and in cell lines, using a cassette containing a second cis-cleaving hammerhead ribozyme positioned 3' of the trans-cleaving hammerhead or hairpin ribozyme. Cis-cleavage could be detected readily in transgenic mice, demonstrating in vivo release of the desired short trans-cleaving ribozyme transcript with a defined 3'-end. In transgenic organs, all cis-cleavage products containing a hairpin ribozyme were found at significantly higher steady-state levels than products containing a hammerhead ribozyme. Furthermore, an organ difference - kidney > liver > lung > spleen - regarding steady-state levels of both 5' and 3' cleavage products was found. In pools of stably transfected human T cells (HUT78), the efficacy of the 3' cis-cleavage was found to affect both the steady-state level and the antiviral efficiency of a trans-cleaving hairpin ribozyme targeting HIV-1. Insertion of a point mutation, efficiently inhibiting the cis-cleavage mechanism, led to higher overall steady-state levels of the noncleaved full-length transcript but, at the same time, also abolished the hairpin ribozyme protection against HIV-1 infection. We conclude that the cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and that it has a strong impact on trans-targeting efficiency.
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