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Downregulation of c-Ki-ras promoter activity by triplex-forming oligonucleotides endogenously generated in human 293
S Cogoi1, C Suraci, E Del Terra
1Department of Biomedical Sciences and Technologies, School of Medicine, Udine, Italy.
Abstract:
Exogenous triplex-forming oligodeoxynucleotides (TFO) have the capacity to modulate in vivo the expression of individual genes. As the administration of TFO to cells is not without problems, we analyzed the possibility of generating them directly in the cell, using specific expression vectors. We constructed three vectors, mU6-GA, mU6-CA, and mU6-CT, that direct the synthesis in human 293 cells of 76-mer CU, GU, and AG motif TFO (rTFO) potentially capable of binding to a critical poly (R x Y) sequence contained in the promoter of the Ki-ras proto-oncogene. The ability of the CU, GU, and AG motif rTFO to interact with the double helix of the c-Ki-ras target was investigated in vitro by footprinting and band-shift experiments, using both synthetic and endogenously synthesized oligoribonucleotides. The human 293 cells were transfected with DNA mixtures containing a plasmid, which bears the reporter chloramphenicol acetyltransferase (CAT) gene downstream from the c-Ki-ras promoter (pKRS-413), as well as an rTFO-generating vector (mU6-GA, mU6-CA, or mU6-CT). As control, the cells were transfected with DNA mixtures containing vector mU6-C1 or mU6-C2. These generated transcripts unable to form triple helices with the poly (R x Y) sequence of the c-Ki-ras promoter. Intracellular synthesis of the 76-mer CU, GU, and AG rTFO by mU6-GA, mU6-CA, and mU6-CT was checked by Northern blot hybridization. Through beta-gal and CAT ELISA immunoassays, we found that the 293 cells transfected with either mU6-GA, mU6-CA, or mU6-CT showed a significant inhibition of CAT expression compared with cells transfected with control plasmids mU6-C1 or mU6-C2. The results of five separate transient transfection experiments showed that endogenous GU and AG rTFO, generated by mU6-CA and mU6-CT, produce, respectively, 40% (+/- 4% SE) and 47% (+/- 8% SE) CAT inhibition, whereas CU rTFO, generated by mU6-GA, produces 38% (+/- 7% SE) CAT inhibition. In conclusion, this study suggests that it is possible to downregulate the expression of an individual gene through the use of recombinant vectors encoding the information for the intracellular synthesis of short triplex-forming RNA strands.
Insights
Researchers developed expression vectors to generate triplex-forming oligodeoxynucleotides (TFO) inside cells. This approach successfully downregulated c-Ki-ras proto-oncogene expression by up to 47% in human cells.
Area of Science:
- Molecular Biology
- Gene Regulation
- Oligonucleotide Therapeutics
Background:
- Exogenous triplex-forming oligodeoxynucleotides (TFO) can modulate gene expression in vivo.
- Direct intracellular synthesis of TFOs presents an alternative to exogenous administration, potentially overcoming delivery challenges.
Purpose of the Study:
- To investigate the feasibility of generating triplex-forming RNA oligonucleotides (rTFO) intracellularly using specific expression vectors.
- To assess the ability of these endogenously synthesized rTFOs to downregulate the expression of the c-Ki-ras proto-oncogene.
Main Methods:
- Construction of three expression vectors (mU6-GA, mU6-CA, mU6-CT) for synthesizing 76-mer CU, GU, and AG motif rTFOs in human 293 cells.
- In vitro validation of rTFO-target interaction using footprinting and band-shift assays.
- Transient transfection of 293 cells with reporter plasmids (c-Ki-ras promoter driving CAT gene) and rTFO-generating vectors, with controls.
- Assessment of intracellular rTFO synthesis via Northern blot and gene expression inhibition via CAT ELISA.
Main Results:
- Northern blot confirmed intracellular synthesis of CU, GU, and AG rTFOs.
- Transfection with rTFO-generating vectors resulted in significant inhibition of chloramphenicol acetyltransferase (CAT) expression driven by the c-Ki-ras promoter.
- Endogenous GU and AG rTFOs achieved 40% and 47% CAT inhibition, respectively, while CU rTFOs achieved 38% inhibition.
Conclusions:
- This study demonstrates the successful intracellular synthesis of functional rTFOs using recombinant expression vectors.
- The findings support the potential of this approach for targeted gene downregulation, offering a novel strategy for gene therapy and molecular biology research.