Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions.
Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions.
复制标题
DOI：
10.1093/nar/28.18.e83
复制
发表时间：
2000-09-15
影响因子：
14.9
通讯作者：
Lohse, P A
中科院分区：
文献类型：
--
作者：
Kurz, M;Gu, K;Lohse, P A
我们描述了一种新型mRNA模板的合成及其在mRNA-蛋白质融合物制备中的应用。使用光诱导的嘌呤霉素交联反应将含嘌呤霉素的寡核苷酸连接到mRNA模板的3 '端。发现光交联模板在兔网织红细胞裂解物中进行有效的mRNA-蛋白质融合形成。随后用携带不同长度和化学组成的嘌呤霉素接头的模板测试融合形成。具有多个三甘醇磷酸酯结构单元的短接头允许在宽范围的盐条件下最有效的融合形成。本发明的方法简化了mRNA-蛋白质融合体的制备，从而显著加速了体外蛋白质进化过程，该过程涉及融合体产生和选择的重复循环。
We describe the synthesis of a novel type of mRNA template and its use in the preparation of mRNA-protein fusions. A light-induced psoralen crosslinking reaction was used to attach a puromycin-containing oligonucleotide to the 3'-end of an mRNA template. The photo-crosslinked template was found to undergo efficient mRNA-protein fusion formation in rabbit reticulocyte lysate. Fusion formation was subsequently tested with templates carrying puromycin linkers of different length and chemical composition. Short linkers with multiple triethyleneglycol phosphate building blocks allowed the most efficient fusion formation under a wide range of salt conditions. The present method simplifies the preparation of mRNA-protein fusions and thus significantly accelerates the in vitro protein evolution procedure which involves repetitive cycles of fusion production and selection.