ABSTRACT
Eighteen peptide-oligonucleotide phosphorothioate conjugates were prepared in good yield and thoroughly characterized with electrospray ionization mass spectra. When applied to the living cells, conjugates exhibiting membrane translocation and nuclear localization properties displayed efficient intracellular penetration but failed to show any serious antisense effect. Studies on the intracellular distribution of the fluorescein-labeled conjugates revealed their trapping in endosomes.
Subject(s)
Cell Nucleus/chemistry , Oligonucleotides/chemistry , Oligopeptides/chemistry , Organothiophosphorus Compounds/chemistry , Cell Membrane/chemistry , Cross-Linking Reagents , Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescein-5-isothiocyanate , Gene Expression/genetics , Hydrolysis , Luciferases/biosynthesis , Luciferases/genetics , Mass Spectrometry , Microscopy, Confocal , Oligonucleotides/chemical synthesis , Oligonucleotides/metabolism , Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides, Antisense/chemistry , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Organothiophosphorus Compounds/chemical synthesis , Organothiophosphorus Compounds/metabolism , Ribonuclease HABSTRACT
Several circular oligonucleotides were synthesized and characterized by electrospray ionization mass spectrometry. Experiments on termination of primer extension catalysed by DNA polymerases, Klenow fragment and Tth have demonstrated that a double helix forming circular 2'-deoxyribooligomer containing a 25mer sequence complementary to the target single-stranded DNA along with a 34mer random mismatching stretch appears to be a potent inhibitor of replication in vitro. Studies on inhibition of luciferase gene expression in a cell-free transcription-translation system have shown that a duplex forming circular 2'-deoxyribooligonucleotide containing a 25mer sequence complementary to the target mRNA and a 14mer random mismatching stretch can serve as an effective antisense compound as a standard linear complementary oligomer. Features of double helix forming circular oligonucleotides composed of 2'-deoxyribonucleosides seem to be useful for the design of new antigene and antisense agents.
