Department of Biotechnology, Ocean University of China, Qingdao, Shandong, People's Republic of China.
PLoS One. 2013 Jul 4;8(7):e67558. doi: 10.1371/journal.pone.0067558. Print 2013.
Antisense oligonucleotides (ASODNs) have been widely used as an important tool for regulating gene expression, and developed into therapeutics. Natural ODNs are susceptible to nuclease degradation, nucleic acid analogues, however, have less side effects, stronger stability and more potent activities. Large-scale de novo synthesis of a certain oligonucleotide has been very difficult and costly. In a previous preliminary study, we developed the polymerase-endonuclease amplification reaction (PEAR) for amplification and large-scale preparation of natural antisense ODNs. Here we extended the method in preparation of a widely used modified oligonucleotide with 5'-O-(1-Thiotriphosphate) modifications. Using electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection, the purity of the PEAR product was measured as high as 100.0%. Using PEAR a large amount of a specific oligonucleotide can be produced starting from a small amount of synthetic seeds. It is suggested that PEAR can be a useful tool for large-scale production of modified oligonucleotides.
反义寡核苷酸 (ASODNs) 已被广泛用作调节基因表达的重要工具，并已发展成为治疗药物。天然 ODN 容易被核酸酶降解，而核酸类似物则副作用较小、稳定性更强、活性更高。大规模从头合成特定的寡核苷酸一直非常困难且昂贵。在之前的初步研究中，我们开发了聚合酶内切酶扩增反应 (PEAR)，用于扩增和大规模制备天然反义 ODN。在这里，我们将该方法扩展到制备广泛使用的带有 5′-O-(1-硫代三磷酸)修饰的修饰寡核苷酸。使用电喷雾电离液相色谱质谱 (ESI/LC/MS) 检测，PEAR 产物的纯度高达 100.0%。使用 PEAR，可以从小量合成的种子开始大量生产特定的寡核苷酸。因此，PEAR 可以成为大规模生产修饰寡核苷酸的有用工具。
