Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking.
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.
Intody
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Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
Perkins
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Wilson
J H
JH
Wensel
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TG
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D004249:DNA Damage / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000528:radiation effects; D005363:Ficusin / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry; D005453:Fluorescence; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D017319:Photosensitizing Agents / Q000378:metabolism / Q000494:pharmacology* / Q000528:radiation effects / Q000627:therapeutic use; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000628:therapy; D012243:Rhodopsin / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D017319:Photosensitizing Agents; D012333:RNA, Messenger; C051802:triplex DNA; D004247:DNA; D012243:Rhodopsin; D005363:Ficusin