The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells.
Chimeric oligodeoxynucleotides with terminal nonionic methylphosphonate analogue sections and internal phosphodiester regions offer several advantages as antisense effectors over either structure alone. These include enhanced biological stability relative to all-phosphodiester molecules, increased activity in directing ribonuclease H mediated destruction of target RNA, increased specificity and reduced non-specific toxicity. However, another important parameter, the ability of these molecules to enter intact mammalian cells, has not previously been investigated. Therefore, oligodeoxynucleotides were tagged at their 5'-termini with fluorescein reporter groups and a detailed study of uptake kinetics in human leukaemia MOLT-4 cells undertaken by calibrated flow cytometry. Baseline measurements with all-phosphodiester and all-methylphosphonate molecules confirmed that uptake of oligodeoxynucleotides by intact cells is a highly inefficient process. The kinetic data were in agreement with previous reports of mechanisms of cell uptake involving receptor mediated endocytosis in the case of phosphodiester molecules and simple diffusion for methylphosphonates. Chimeric oligodeoxynucleotides exhibited saturable cell uptake kinetics similar to all-phosphodiester oligodeoxynucleotides, suggesting that uptake was receptor-mediated and distinct from concentration-dependent uptake of all-methylphosphonate molecules. Similarly, chimeric molecules were apparently confined to the endosomal compartment within cells. These results imply that reversible masking of the negative charges of the phosphodiester sections of chimeric oligodeoxynucleotides may be required to change the uptake mechanism back to simple diffusion and allow intracellular delivery to the site of the target RNA.
Spiller
D G
DG
Department of Biochemistry, University of Liverpool, UK.
Tidd
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D005434:Flow Cytometry; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides