Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells
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- 作者：Faria ; Marcella ; Wood ; Chris D. ; White ; Mike R.H. ; Hé ; lè ; ne ; Claude ; Giovannangeli ; Carine
- 关键词：phosphoramidate oligonucleotide ; psoralen ; transcription elongation ; triplex ; PPT ; Pso- ; psoralen-conjugated ; TFO ; triple helix-forming oligonucleotide ; PPT ; polypurine tract ; po ; phosphodiester ; np ; N3&prime ; -P5&prime ; -phosphoramidate ; PGK ; phosphoglycer
- 刊名：Journal of Molecular Biology
- 出版年：2001
- 出版时间：February 9, 2001
- 年：2001
- 卷：306
- 期：1
- 页码：15-24
- 全文大小：297 K
文摘
Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3′-P5′ phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple helix-forming oligonucleotides (Pso-TFOs) can additionally be photo-induced to become irreversibly bound to their targeted DNA sequence. Here, we have examined the ability of various 15-mer phosphoramidate TFOs targeted to the HIV-1 polypurine tract (PPT) sequence to prevent transcription elongation in cell cultures; the PPT sequence has been cloned in the transcribed region of a reporter firefly luciferase gene (luc) and transient expression experiments performed. We show that the level of transcription inhibition of the reporter gene in cells perfectly correlates with the amount of covalent triplex at the PPT site. The efficacy of non-covalent triplexes (either omitting the irradiation step with the psoralen conjugate, or using the unsubstituted oligonucleotide) has been studied in our expression system; the oligonucleotides were introduced into living cells by cationic lipid-mediated delivery or directly into the cell nucleus by microinjection. This experimental approach allowed us to evaluate the intrinsic activity of triplexes as transcriptional inhibitors; transcription elongation was inhibited in cells in a sequence-dependent and concentration-dependent manner.