Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli
Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli
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DOI：
10.1128/aac.49.1.249-255.2005
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发表时间：
2005-01-01
影响因子：
4.9
通讯作者：
Geller, BL
中科院分区：
文献类型：
--
作者：
Deere, J;Iversen, P;Geller, BL
磷酸二酰胺吗啉寡聚物 (PMO) 是合成 DNA 类似物，能够以序列依赖性方式抑制基因表达。测试了不同长度（7 至 20 个碱基）的 PMO 对大肠杆菌中荧光素酶表达的抑制作用。较短的 PMO 通常比较长的 PMO 对荧光素酶的抑制更大。相反，在细菌无细胞蛋白质合成反应中，较长的 PMO 与较短的 PMO 具有相同或更多的抑制作用。与荧光素酶起始密码子周围区域互补的重叠等距（10 个碱基）PMO 在细菌无细胞蛋白表达反应中受到不同程度的抑制。最大抑制不需要在 PMO 中包含反起始密码子。除一种靶向核糖体结合位点的 PMO 外，靶向荧光素酶 mRNA 内 5' 非翻译区或 3' 编码区的 PMO 不会产生抑制作用。荧光素酶表达的抑制与 PMO 靶向的 mRNA 区域的预测二级结构负相关，但与靶向区域的 C+G 含量不相关。通过使用针对 acpP（生存所需的基因）的 PMO（6 至 20 个碱基）证实了 PMO 长度和位置的影响。由于根据之前在真核生物中的结果，类似 11 个碱基的 PMO 的抑制是出乎意料的，因此我们在 HeLa 细胞和网织红细胞无细胞蛋白质合成反应中测试了 11 个碱基的 PMO。 11 碱基 PMO 显着抑制 HeLa 细胞中的荧光素酶表达，但效果比 20 碱基 PMO 的抑制要小。在无网织红细胞反应中，存在更长的 PMO 抑制更多的趋势。这些研究表明原核生物的 PMO 设计策略与真核生物的靶标有很大不同。
Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5' nontranslated or 3' coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C+G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability. Because inhibition by PMOs of similar to11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.