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

DOI
Publisher DOI
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‘M concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction of 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 tricyclo-DNA for nuclear antisense applications. Morover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.
Date Issued
2004
Publication Type
Article
Subject(s)
Subjects
analysis
Antisense
backbone
Cells
cellular
Cyclophilin A
distribution
double
Fluorescein
fluorescence
HeLa
Hela Cells
Hiv-1
LNA
oligonucleotide
Oligonucleotides
properties
Protein
reduction
REPLICATION
site
Transfection
tricyclo-DNA
Language(s)
en
Author(s)
Ittig, D.
Additional Credits
Journal
Nucleic acids research
Publisher
Oxford University Press
ISSN
0305-1048
Access(Rights)
open.access
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