A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest.
A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest.
复制标题
一种筛选对 mRNA 翻译停滞有效的反义寡脱氧核糖核苷酸的方法。
DOI：
10.1093/oxfordjournals.jbchem.a021231
复制
发表时间：
1996
影响因子：
2.7
通讯作者：
L. Au
中科院分区：
文献类型：
--
作者：
T. Chen;S. B. Lin;J. Wu;K. Choo;L. Au
建立了转录和翻译偶联的网织红细胞裂解物系统，用于快速筛选反义寡脱氧核糖核苷酸 (ODN)，以确定哪些对 mRNA 翻译抑制最有效。在 T7（或 SP6）RNA 聚合酶、RNase H 和待测反义 ODN 存在的情况下，将含有 T7（或 SP6）启动子控制下的靶 cDNA 的质粒添加到裂解液系统中。转录和翻译是在单管反应中完成的。根据通过凝胶电泳和放射自显影测量的从头合成的[35S]-甲硫氨酸或[35S]半胱氨酸标记的靶蛋白的量来评估反义ODN引起的翻译停滞。确定了该系统的特性和用于反义 ODN 筛选的最佳反应条件。我们的方法比其他体外筛选方法更简单、更快速。
A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [35S]-methionine or [35S]cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.