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Comparative Study
. 2004 Jan 15;32(1):346-53.
doi: 10.1093/nar/gkh187. Print 2004.

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA

Damian Ittig  1 , Songkai Liu, Dorte Renneberg, Daniel Schümperli, Christian J Leumann
Affiliations
Comparative Study

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA

Damian Ittig et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1
Figure 1
Molecular structures of tc-DNA and LNA in comparison to DNA.
Figure 2
Figure 2
Schematic representation of the splicing pattern of CyPA pre-mRNA. The ball at tc6 or lna6 represents the fluorescein label.
Figure 3
Figure 3
RT–PCR analysis of CyPA pre-mRNA splicing in HeLa cells in the presence of varying concentrations (0.05, 0.1 and 0.2 µM) of antisense tc-oligonucleotides (lanes 1–12). Lane 13 (control): RNA from untreated cells.
Figure 4
Figure 4
Quantitative evaluation of full-length CyPA mRNA after treatment with tc-oligonucleotides. Values are normalized averages of two independent experiments. Control: RNA from untreated cells.
Figure 5
Figure 5
RT–PCR analysis of CyPA pre-mRNA splicing in HeLa cells in the presence of varying concentrations (0.05, 0.1 and 0.2 µM) of antisense lna-oligonucleotides (lanes 1–12). Lane 13 (control): RNA from untreated cells.
Figure 6
Figure 6
Quantitative evaluation of full-length CyPA mRNA after treatment with lna-oligonucleotides. All values are normalized averages with standard deviations from three independent experiments. Control: RNA from untreated cells.
Figure 7
Figure 7
Western blot, demonstrating the down-regulation of CyPA protein by tc4. CyPA protein was probed with polyclonal anti-CyPA antibodies. A polyclonal anti-actin antibody was used for loading control.
Figure 8
Figure 8
Fluorescence microscopic images of the cellular distribution of tc6 (left) and lna6 (right) in HeLa cells (top: phase contrast image; center: UV image; and bottom: overlay of both images).
See this image and copyright information in PMC

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