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Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking
Z Intody1, B D Perkins, J H Wilson
1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.
Abstract:
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.
Insights
Triplex-forming oligodeoxyribonucleotides (TFOs) effectively inhibit gene expression by causing DNA damage. This method shows promise for treating genetic disorders like autosomal dominant retinitis pigmentosa.
Area of Science:
- Molecular Biology
- Genetics
- Oligonucleotide Therapeutics
Background:
- Autosomal dominant retinitis pigmentosa is a blinding disorder caused by mutations in the human rhodopsin gene.
- Gene silencing strategies are needed to treat dominant genetic disorders.
Purpose of the Study:
- To investigate the efficacy of psoralen-modified triplex-forming oligodeoxyribonucleotides (TFOs) in inhibiting the expression of the human rhodopsin gene.
- To assess the potential of TFOs for treating genetic disorders.
Main Methods:
- Psoralen-modified TFOs were designed to target the human rhodopsin gene.
- A plasmid containing a rhodopsin-EGFP fusion gene was transfected into HT1080 cells.
- UVA irradiation was used to induce photoadducts, blocking RNA polymerase progression.
- Gene expression levels were analyzed using Northern blots and fluorescence microscopy.
Main Results:
- Single TFO treatments reduced rhodopsin-GFP expression by 70-80%; combined treatment reduced it by 90%.
- Control gene expression remained unaffected, demonstrating specificity.
- TFO-induced photoadducts resulted in truncated transcripts and sustained gene silencing over 72 hours.
- Inhibition occurred even when photoadducts were formed intracellularly.
Conclusions:
- Psoralen-modified TFOs efficiently and specifically inhibit rhodopsin gene transcription.
- This DNA-damaging approach offers a promising strategy for gene silencing in dominant genetic disorders.
- The observed lack of repair suggests long-lasting therapeutic effects.