Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA)

Rongbin Ge, Mathias G. Svahn, Oscar E. Simonson, Abdalla J. Mohamed, Karin E. Lundin, C. I.Edvard Smith

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)101-109
Number of pages9
JournalJournal of Gene Medicine
Volume10
Issue number1
DOIs
StatePublished - Jan 2008

Keywords

  • Locked nucleic acid
  • RNA polymerase III
  • Transcription
  • shRNA

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