Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.
The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA-binding proteins can be inhibited by the use of double-stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN-kappaB) and circular (CDODN-kappaB) dumbbell DNA oligonucleotides containing a NF-kappaB binding site to analyze the inhibition of the NF-kappaB-dependent activation of the human immunodeficiency virus type-1 (HIV-1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF-kappaB sequence were found to compete with the native strand for NF-kappaB binding. The circular dumbbell and double-stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF-kappaB binding proteins, while the nicked NF-kappaB dumbbell was a less effective competitor. In Jurkat T-cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV-NL4-3 Luc. The CDODN-kappaB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN-kappaB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non-specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti-HIV-1 or other therapeutic effects.
Hosoya
T
T
Department of Industrial Chemistry, Chiba Institute of Technology, Tsudanuma, Narashino, Chiba, Japan.
Takeuchi
H
H
Kanesaka
Y
Y
Yamakawa
H
H
Miyano-Kurosaki
N
N
Takai
K
K
Yamamoto
N
N
Takaku
H
H
D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D019169:Jurkat Cells; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection
D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D008156:Luciferases