Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA
AUTOR(ES)
Ittig, Damian
FONTE
Oxford University Press
RESUMO
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 µM 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.
