Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells.
Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.
Hoque
A T
AT
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
Sedelnikova
O A
OA
Luu
A N
AN
Swaim
W D
WD
Panyutin
I G
IG
Baum
B J
BJ
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D051402:Aquaporin 5; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007457:Iodine Radioisotopes / Q000737:chemistry; D008565:Membrane Proteins; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D051381:Rats; D014162:Transfection
C496040:Aqp5 protein, rat; D051402:Aquaporin 5; D020346:Aquaporins; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D009841:Oligonucleotides; C051802:triplex DNA; D011094:Polyethyleneimine; D004247:DNA