Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease H with high specificity.
A previous report has demonstrated that normal phosphodiester oligodeoxynucleotides could direct extensive non-targeted ribonuclease (RNase) H-dependent effects, and that greatly enhanced specificity could be achieved upon methylphosphonodiester substitution of terminal phosphodiester residues. In this report, we extend our previous observations to show that phosphorothioate oligodeoxynucleotides also direct substantial inappropriate RNase H-mediated hydrolysis of non-targeted RNA. Chimeric methylphosphonodiester/phosphodiesters were found to be capable of efficiently directing RNase H when the central phosphodiester section was reduced to just two contiguous internucleoside linkages. Furthermore, cleavage of non-target RNA sites was found to be undetectable, or minimal in extent, when RNase H was directed by such chimeras. In addition, we show that analogue structures which contain three, or fewer, phosphodiester residues in otherwise methylphosphonodiester molecules were imported into cells via the comparatively more efficient route taken by methylphosphonates, rather than by receptor-mediated endocytosis, which is generally characteristic for polyanionic structures. Evidence is presented that the primary process responsible for enhanced uptake is an active mechanism. Nevertheless, a proportion of the applied oligodeoxynucleotide analogues, which demonstrate augmented uptake, appear to have penetrated into the cytoplasmic cellular compartment. The present results suggest that chimeric molecules of the type we describe here may show considerable utility as antisense effectors due to their increased cellular import, access to the intracellular compartments, and their highly efficient and specific direction of RNase H.
Department of Biochemistry, University of Liverpool, UK.
R V
RV
Giles
D G
DG
Spiller
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Tidd
D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003720:Densitometry; D005434:Flow Cytometry; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000493:pharmacokinetics* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000302:isolation & purification; D016914:Ribonuclease H / Q000187:drug effects*; D012680:Sensitivity and Specificity; D013873:Thionucleotides / Q000138:chemical synthesis / Q000493:pharmacokinetics* / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D012334:RNA, Neoplasm; D013873:Thionucleotides; D016914:Ribonuclease H