Nucleic Acids Research, 2000, Vol. 28, No. 21 4283-4290
© 2000 Oxford University Press
Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA
To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin–EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin–GFP expression by 70–80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen–TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin–EGFP. We conclude that directing DNA damage with psoralen–TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene.
* To whom correspondence should be addressed. Tel: +1 713 798 6994; Fax: +1 713 796 9438; Email: twensel@bcm.tmc.edu Permanent address: Zsofia Intody, Department of Ophthalmology No. 1, Semmelweis University of Medicine, Budapest, Hungary Present address: Brian D. Perkins, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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