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Sequence specific inhibition of in vitro translation of mutated or normal ras p21
1Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
Abstract:
Antisense methylphosphonate-modified oligomers (ONMP) complementary to 8 nucleotides spanning the twelfth amino acid codon of human c-Ha-ras have been synthesized to explore their inhibitory effect on ras p21 translation. The ONMP Ras 0, perfectly complementary to the specific target region in normal c-Ha-ras, inhibits cell-free translation of p21 from a normal c-Ha-ras mRNA template in a dose-dependent manner. At 200uM Ras 0, p21 translation is inhibited by almost 90% in a rabbit reticulocyte lysate; at 100uM, p21 is reduced by over 60%. Ras I and Ras II contain, internally, a single and double base mismatch, respectively. The inhibitory activity of these ONMPs is reduced in proportion to the number of mismatches. When the target mRNA encodes the activated c-Ha-ras differing by a single nucleotide at the twelfth amino acid codon from normal c-Ha-ras, the magnitude of the inhibitory effect of Ras I increased significantly because Ras I is now perfectly complementary to its target mRNA. In turn, Ras 0, now only partially complementary, is considerably less effective at the same concentrations. Therefore, the inhibitory effect is exquisitely sensitive to the extent of the sequence complementarity between the antisense ONMP and the targeted mRNA.
Insights
Antisense methylphosphonate-modified oligomers (ONMP) effectively inhibit ras p21 translation. Their inhibitory effect is highly sensitive to sequence complementarity with the target mRNA, with perfect matches yielding the strongest inhibition.
Area of Science:
- Molecular Biology
- Antisense Technology
- Oncogene Research
Background:
- The ras proto-oncogene family plays a crucial role in cell signaling and cancer development.
- Targeting oncogene translation is a potential therapeutic strategy for cancer.
- Antisense oligonucleotides offer a means to specifically inhibit gene expression.
Purpose of the Study:
- To synthesize and evaluate antisense methylphosphonate-modified oligomers (ONMP) for their ability to inhibit human c-Ha-ras p21 translation.
- To investigate the impact of sequence complementarity on the inhibitory efficacy of ONMPs.
Main Methods:
- Synthesis of antisense methylphosphonate-modified oligomers (ONMP) targeting the twelfth amino acid codon of human c-Ha-ras mRNA.
- In vitro cell-free translation assays using rabbit reticulocyte lysate.
- Dose-response analysis of ONMP inhibition.
- Evaluation of ONMPs with varying degrees of complementarity to target mRNA.
Main Results:
- A perfectly complementary ONMP (Ras 0) significantly inhibited cell-free translation of normal c-Ha-ras p21 in a dose-dependent manner (up to 90% inhibition at 200uM).
- ONMPs with single (Ras I) or double (Ras II) base mismatches showed reduced inhibitory activity, proportional to the degree of mismatch.
- Ras I demonstrated significantly increased inhibition of an activated c-Ha-ras mRNA (with a single nucleotide difference) due to perfect complementarity, while Ras 0's efficacy decreased.
Conclusions:
- Antisense methylphosphonate-modified oligomers can effectively inhibit ras p21 translation.
- The inhibitory potency of ONMPs is critically dependent on the precise sequence complementarity between the oligomer and its target mRNA.
- These findings highlight the potential of sequence-specific antisense technology for targeting oncogene expression.