Duroux I, Godard G, Boidot-Forget M, Schwab G, Hélène C, Saison-Behmoaras T
Laboratoire de Biophysique, INSERM U.201, CNRS URA 481, Muséum National d'Histoire Naturelle, Paris, France.
Nucleic Acids Res. 1995 Sep 11;23(17):3411-8. doi: 10.1093/nar/23.17.3411.
Antisense oligodeoxynucleotides targeted to Ha-ras mRNA have been designed to discriminate between the codon 12-mutated oncogene and the normal proto-oncogene. An in vitro assay using two different sources of RNase H (rabbit reticulocyte lysates and nuclear extract from HeLa cells) was used to characterize oligonucleotide binding to normal and mutated Ha-ras mRNA. Short oligonucleotides (12- or 13mers) centered on the mutation had a very high discriminatory efficiency. Longer oligonucleotides (16mers) did not discriminate efficiently between the mutated and the normal mRNA. We have tested the efficacy of dodecanucleotides to induce RNase H cleavage of the full-length mRNA, moving the target sequence from the loop to the stem region which is formed in the vicinity of mutated codon 12. The most selective oligonucleotides were centered on the mutation which is located near the junction between the loop and stem regions even though they were less efficient at inducing RNase H cleavage than those targeted to the loop region. The 12mer antisense oligonucleotide with the highest discriminatory power was selected for cell culture studies. This oligonucleotide inhibited the proliferation of a human cell line which had been transformed with the mutated Ha-ras gene (HBL100ras1) but had no effect on the parental cell line which was transfected with the vector DNA (HBL 100neo) and expressed only the normal Ha-ras gene. Growth inhibition of HBL100ras1 cells was associated with specific ablation of targeted Ha-ras mRNA as shown by RT-PCR. These results show that 'in vitro' evaluation using an RNase H assay allowed us to select an antisense oligonucleotide which elicited a selectivity towards point-mutated Ha-ras mRNA when added at 10 microM concentration to the culture medium of cells expressing wild type and mutated Ha-ras mRNA.
针对Ha-ras mRNA设计的反义寡脱氧核苷酸旨在区分密码子12突变的癌基因和正常原癌基因。使用两种不同来源的RNase H（兔网织红细胞裂解物和HeLa细胞核提取物）进行体外试验，以表征寡核苷酸与正常和突变的Ha-ras mRNA的结合情况。以突变位点为中心的短寡核苷酸（12或13聚体）具有非常高的鉴别效率。较长的寡核苷酸（16聚体）不能有效区分突变型和正常mRNA。我们测试了十二聚体诱导全长mRNA的RNase H切割的效果，将靶序列从环区移至在突变密码子12附近形成的茎区。最具选择性的寡核苷酸以位于环区和茎区交界处附近的突变位点为中心，尽管它们诱导RNase H切割的效率低于靶向环区的寡核苷酸。选择具有最高鉴别能力的12聚体反义寡核苷酸进行细胞培养研究。该寡核苷酸抑制了用突变的Ha-ras基因（HBL100ras1）转化的人细胞系的增殖，但对用载体DNA转染并仅表达正常Ha-ras基因的亲本细胞系（HBL 100neo）没有影响。如RT-PCR所示，HBL100ras1细胞的生长抑制与靶向的Ha-ras mRNA的特异性缺失有关。这些结果表明，使用RNase H试验进行的“体外”评估使我们能够选择一种反义寡核苷酸，当以10 microM的浓度添加到表达野生型和突变型Ha-ras mRNA的细胞培养基中时，该寡核苷酸对点突变的Ha-ras mRNA具有选择性。
