Cellular uptake properties of a 2'-amino/2'-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA.
Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.
Fell
P L
PL
Pharmaceutical Sciences Institute, Aston University, Birmingham, U.K.
Hudson
A J
AJ
Reynolds
M A
MA
Usman
N
N
Akhtar
S
S
D001483:Base Sequence; D001667:Binding, Competitive; D001692:Biological Transport; D017124:Conserved Sequence; D004247:DNA / Q000494:pharmacology; D016264:Dextran Sulfate / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D066246:ErbB Receptors / Q000096:biosynthesis*; D005909:Glioblastoma; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013095:Spermidine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013873:Thionucleotides; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D010761:Phosphorus Radioisotopes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D013873:Thionucleotides; D006493:Heparin; D004247:DNA; D016264:Dextran Sulfate; D066246:ErbB Receptors; D013095:Spermidine