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Evaluation of N-ras oncogene anti-sense, sense and nonsense sequence methylphosphonate oligonucleotide analogues
D M Tidd1, P Hawley, H M Warenius
1Cancer Research Campaign Department of Radiation Oncology, University of Liverpool.
Abstract:
We have investigated the potential for using anti-sense non-ionic methylphosphonate oligonucleotide analogues to study the relationship between oncogene expression and maintenance of the transformed phenotype in malignant cells. Our results confirmed that the methylphosphonates are resistant to biochemical degradation and are devoid of non-specific toxicity towards cultured human HT29 cells. At low temperature (less than 5 degrees C) both N-ras anti-sense and nonsense analogue 9-mers formed 1:1 complexes in solution with an N-ras sense phosphodiester oligodeoxynucleotide 20-mer, but these were largely dissociated at 25 degrees C. Only a fraction (10-20%) of the anti-sense molecules formed stable sequence specific hybrids (Tm 34 degrees C) with the 20-mer. The biological activity of the oligonucleotide analogues was tested in cell culture at 37 degrees C using T15 cells, a line of NIH 3T3 cells transfected with multiple copies of the human N-ras oncogene under control of the glucocorticoid inducible MMTV promoter. On balance the N-ras anti-sense methylphosphonate 9-mer (20-80 microM) had no effect on these cells. In only one of five experiments was an apparent reduction in dexamethasone-induced p21N-ras protein accumulation observed in the presence of the oligonucleotide analogue. Also without effect was an anti-sense 20-mer consisting of a phosphodiester sequence bounded by two methylphosphonate linkages at each end (25-50 microM in culture media; 4.8 microM by microinjection). We conclude from these experiments that, in order to achieve pronounced effects on oncogene expression, it may be necessary to use longer anti-sense methylphosphonate chains, affinity purified for their ability to hybridize with the target sequences.
Insights
Methylphosphonate oligonucleotides show resistance to degradation and low toxicity. However, short N-ras anti-sense sequences did not effectively inhibit oncogene expression in cell culture, suggesting longer chains are needed.
Area of Science:
- Molecular Biology
- Cancer Research
- Oligonucleotide Therapeutics
Background:
- Oncogene expression is crucial for maintaining the transformed phenotype in malignant cells.
- Anti-sense oligonucleotides offer a potential strategy to modulate oncogene expression.
- Methylphosphonate analogues are investigated for enhanced stability and reduced toxicity.
Purpose of the Study:
- To evaluate the efficacy of anti-sense methylphosphonate oligonucleotides in suppressing oncogene expression.
- To assess the relationship between oncogene expression and the transformed cellular phenotype.
- To determine the suitability of methylphosphonate analogues for anti-sense applications.
Main Methods:
- Synthesis and characterization of N-ras anti-sense and nonsense methylphosphonate 9-mer analogues.
- Hybridization studies with N-ras sense phosphodiester oligodeoxynucleotide at varying temperatures.
- Assessment of biological activity in T15 cells (NIH 3T3 transfected with human N-ras oncogene) at 37°C.
- Measurement of p21N-ras protein accumulation in response to oligonucleotide treatment.
Main Results:
- Methylphosphonate analogues demonstrated resistance to biochemical degradation and low non-specific toxicity in HT29 cells.
- N-ras anti-sense and nonsense 9-mers formed unstable complexes with a 20-mer sense strand, with limited stable hybridization.
- Short N-ras anti-sense methylphosphonate oligonucleotides (9-mers and modified 20-mers) showed no significant effect on N-ras oncogene expression or cellular phenotype in T15 cells.
Conclusions:
- Short anti-sense methylphosphonate oligonucleotides are not effective in inhibiting oncogene expression.
- Achieving pronounced effects on oncogene expression may require longer anti-sense methylphosphonate chains.
- Affinity purification of oligonucleotides for target sequence hybridization is crucial for efficacy.