Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA.
The nuclear antisense properties of a series of tricyclo (tc)-DNA oligonucleotide 9-15mers, targeted against the 3' and 5' splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence- and dose-dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by a factor of at least 4-5. A tc-oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2 microM concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction in CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc-15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc- and LNA- oligonucleotides. The obtained results confirm the power of tc-DNA for nuclear antisense applications. Moreover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.
Ittig
Damian
D
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland.
Liu
Songkai
S
Renneberg
Dorte
D
Schümperli
Daniel
D
Leumann
Christian J
CJ
D017398:Alternative Splicing; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D021984:Cyclophilin A / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D005091:Exons / Q000235:genetics; D015497:HIV-1 / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008055:Lipids; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D020871:RNA Stability; D013816:Thermodynamics; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D022821:RNA Splice Sites; D021984:Cyclophilin A