RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer
RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer
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DOI：
10.1016/s0022-2836(02)00474-6
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发表时间：
2002-07-12
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;Sénamaud-Beaufort, C;Saison-Behmoaras, TE
最近，我们已经表明，肽核酸（PNA）的十三聚体靶向的密码子74，128和149区域的Ha-ras mRNA逮捕翻译延长在体外。我们的数据首次证明，具有针对信使RNA编码区的混合碱基序列的PNA可以阻止翻译机器和多肽链延伸。与PNA十三聚体和Ha-ras mRNA形成的复合物的特性依赖于PNA-mRNA杂交体的稳定性，其不被细胞蛋白或多种变性条件解离。在本研究中，我们表明，较短的PNA，如十二聚体或十一聚体靶向密码子74区逮捕翻译延长体外。13、12和11-mer PNA含有8个连续的嘧啶残基，10-mer PNA含有7个连续的嘧啶残基。在以平行Hoogsteen碱基配对与PNA-RNA双链体结合后，第二PNA的六个胞嘧啶碱基和一个胸腺嘧啶碱基可以形成C（.）G * C+和T（.）A * T三胞胎解链实验显示了两个良好分辨的转变，对应于第三链从核心双链体的解离和在较高温度下双链体的解链。靶27-mer RNA的酶促结构作图揭示了在三聚体、十二聚体、十一聚体和十聚体PNA结合后被破坏的发夹结构。我们表明，RNA上的非键合的核碱基突出端稳定PNA-RNA杂交体，并可能帮助PNA克服RNA靶的稳定二级结构。PNA-RNA双链体和三链体结构的高度稳定性使我们能够使用基质辅助激光解吸/电离飞行时间质谱法鉴定1：1和2：1 PNA-RNA复合物。因此，有可能用短PNA寡核苷酸成功靶向信使RNA结构化区域中的混合序列，所述短PNA寡核苷酸形成可以阻止延长核糖体的双链体和三链体结构。(C)2002爱思唯尔科技有限公司版权所有。
Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C(.)G * C+ and T(.)A * T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes. (C) 2002 Elsevier Science Ltd. All rights reserved.