title: Effects of base modifications on antisense properties of 2'-O-methoxyethyl and PNA oligonucleotides.
abstract: 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.
authors.affiliation: Lineberger Comprehensive Cancer Center & Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA. sazani@molbio.mgh.harvard.edu
authors.forename: Peter
authors.initials: P
authors.lastname: Sazani
authors.forename: Anna
authors.initials: A
authors.lastname: Astriab-Fischer
authors.forename: Ryszard
authors.initials: R
authors.lastname: Kole
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D001483:Base Sequence; D002412:Cations / Q000737:chemistry; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004337:Drug Carriers / Q000493:pharmacokinetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000378:metabolism; D015394:Molecular Structure; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics / Q000633:toxicity; D010078:Oxazines / Q000494:pharmacology; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D012322:RNA Precursors / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D002412:Cations; D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D016376:Oligonucleotides, Antisense; D010078:Oxazines; D020135:Peptide Nucleic Acids; D012322:RNA Precursors; D049452:Green Fluorescent Proteins; C039203:phenoxazine