Cellular uptake, distribution, and stability of 10-23 deoxyribozymes.
The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.
Dass
Crispin R
CR
Johnson and Johnson Research Laboratories, Eveleigh, Australia.
Saravolac
Edward G
EG
Li
Yang
Y
Sun
Lun-Quan
LQ
D001483:Base Sequence; D002384:Catalysis; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D014162:Transfection
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; C402582:RNA-cleaving DNA 10-23