Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Antiviral Res. 2010 Sep;87(3):345-52. doi: 10.1016/j.antiviral.2010.06.003. Epub 2010 Jun 12.
Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2'-5' oligoadenylate synthetases, which synthesize 2'-5' adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2'-5' adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2'-5' adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail. Here, we report a method suitable to produce large amounts of 2-5As of defined lengths in vitro using porcine OAS1 (pOAS) and human OAS2 (hOAS). We have synthesized a broad spectrum of 2-5As at the milligram scale and report an HPLC-purification and characterisation protocol with quantified yield for 2-5A of various lengths.
病毒感染后，几种细胞 2'-5'寡聚腺苷酸合成酶可在宿主细胞中早期检测到双链病毒 RNA，它们合成 2'-5'腺苷酸寡核苷酸，激活细胞 RNA 酶 L，通过自身和非自身 RNA 切割引发早期初级抗病毒反应。用合成的 2'-5'腺苷酸寡核苷酸转染细胞可激活 RNA 酶 L，因此为研究细胞和病毒进入该途径的早期步骤提供了有用的捷径。已可在体外合成特定的 2'-5' 腺苷酸寡核苷酸，但它们的可控合成、纯化和表征尚未详细报道。在这里，我们报告了一种使用猪 OAS1（pOAS）和人 OAS2（hOAS）在体外大量生产特定长度 2-5As 的方法。我们已经在毫克规模上合成了广泛的 2-5As，并报告了一种 HPLC 纯化和表征方案，可定量生成各种长度的 2-5A。
