Effects of base modifications on antisense properties of 2'-O-methoxyethyl and PNA oligonucleotides.

A recently developed antisense splicing assay was used to determine the relative activities of 2'-O-methoxyethoxy (2'-MOE) phosphorothioate oligonucleotides containing base modifications. In the assay, RNase H-inactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an Enhanced Green Fluorescence Protein (EGFP) reporter pre-mRNA stably expressed in HeLa cells. Thus, the extent of EGFP upregulation is proportional to the antisense activity of the tested molecule. The base modifications included C-5 propynyl analogs of uridine and cytidine and phenoxazine and G-clamp analogs of cytosine. Base-modified 2'-MOE oligonucleotides were delivered to the HeLa EGFP-654 test cells by cationic lipid transfection or scrape-loading or without any delivery method (free uptake). When delivered with a cationic lipid, the G-clamp and phenoxazine oligomers showed increases in activity over the unmodified 2'-MOE parent compound. However, when delivered by scrape-loading or without a delivery method, the unmodified oligomer performed best. The results suggest that base modifications do not enhance the free uptake activity of RNase H inactive 2'-MOE oligomers.