Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA).
BACKGROUND
RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule 'Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA.


METHODS
In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed.


RESULTS
The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model.


CONCLUSIONS
Thus, this new form of gene silencer 'Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs.
Ge
Rongbin
R
Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
Svahn
Mathias G
MG
Simonson
Oscar E
OE
Mohamed
Abdalla J
AJ
Lundin
Karin E
KE
Smith
C I Edvard
CI
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D006801:Humans; D007273:Injections, Intramuscular; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009841:Oligonucleotides / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012320:RNA Polymerase III / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D009841:Oligonucleotides; C477371:locked nucleic acid; D004247:DNA; D012320:RNA Polymerase III