Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation
Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation
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DOI：
10.1006/jmbi.1999.3277
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发表时间：
1999-11-26
影响因子：
5.6
通讯作者：
Saison-Behmoaras, TE
中科院分区：
文献类型：
--
作者：
Dias, N;Dheur, S;Saison-Behmoaras, TE
我们以前已经描述了突变选择性反义寡核苷酸的合理设计，针对致癌Ha-ras mRNA的密码子12。为了进一步提高这些未修饰的寡核苷酸的生物学功效，我们研究了三种不同类型的修饰：肽核酸骨架（PNA）、糖修饰（2 ′-O-甲基）和氨基磷酸酯连接（PN）。我们表明，PNA是独特的空间阻断剂，其能力，在体外特异性地抑制Ha-ras mRNA的翻译。PNA RNA杂合物（T-m = 86 ℃）不被细胞蛋白质解离并且抵抗苯酚提取和尿素变性条件，特异性地阻断突变的Ha-ras mRNA的翻译。与野生型Ha-ras mRNA形成具有中心错配的双链体的PNA十三聚体对mRNA翻译几乎没有影响。密码子12位于翻译起始位点附近，PNA在该位置的杂交可能干扰翻译起始复合物的组装。为了测试多肽链延伸是否也可以被阻断，我们将PNA十三聚体靶向74、128和149区域中的密码子。这些PNA形成与靶向密码子12区的PNA形成的双链体同样稳定的双链体（13个中的10个G. C碱基对）。我们发现，PNA-RNA双链体阻断80 S核糖体的进展。因此，有可能通过使用靶向富含G + C的序列的形成寡核苷酸的PNA寡核苷酸来阻止翻译并伴随产生截短的蛋白质。我们的数据首次证明了非共价双链体可以阻止翻译机制和多肽链的延伸。(C)北京：科学出版社.
We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. in order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate Linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA RNA hybrid (T-m = 86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G + C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation. (C) 1999 Academic Press.