Nuclear antisense effects of neutral, anionic and cationic oligonucleotideanalogs

The antisense activity of oligomers with 2 ' -O-methyl (2 ' -O-Me) phosphor othioate, 2 ' -O-methoxyethyl (2 ' -O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing ass ay in which the modified oligonucleotides blocked aberrant and restored cor rect splicing of modified enhanced green fluorescent protein (EGFP) precurs or to mRNA (pre-mRNA), generating properly translated EGFP. In this approac h, antisense activity of each oligomer was directly proportional to up-regu lation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers con firmed validity of the functional assay. The results showed that the free u ptake and the antisense efficacy of neutral morpholino derivatives and cati onic PNA were much higher than that of negatively charged 2 ' -O-Me and 2 ' -O-MOE congeners. The effects of the PNA oligomers were observed to be dep endent on the number Of L-lysine (Lys) residues at the C-terminus. The expe riments suggest that the PNA containing Lys was taken up by a mechanism sim ilar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.