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Hairpin antisense oligonucleotides containing 2'-methoxynucleosides with base-pairing in the stem region at the 3'-end: penetration, localization, and Anti-HIV activity.

Hairpin antisense oligodeoxyribonucleotides containing 2'-methoxynucleosides were more active in the micromolar concentration range than linear and DNA hairpin phosphorothioate oligonucleotides with the same sequence. Furthermore, the abilities of hairpin antisense and random hairpin phosphorothioate oligonucleotides to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-oligomers of the same length or with the same internucleotide modification. Four different target sites (gag, pol, rev, and tat) within the HIV-1 genome were studied with regard to the inhibition of HIV-1 replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of the initiation sequences of gag were most effective. The [32P]-labeled hairpin phosphorothioate oligonucleotide was rapidly assimilated by MOLT-4 cells, whereas the [32P]-labeled hairpin phosphodiester oligonucleotide was not. In MOLT-4 cells treated with the FITC-hairpin phosphorothioate oligonucleotide containing 2'-methoxynucleosides by a confocal laser scanning microscope, diffuse fluorescence was observed in the cytoplasm. Interestingly, fluorescent signals accumulated in the nuclear region of chronically infected MOLT-4/HIV-1 cells after a 60 min incubation.