Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green.
Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factor and malachite green.
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工程化 5S 核糖体 RNA 显示识别血管内皮生长因子和孔雀石绿的适体。
DOI：
10.1002/jmr.917
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
Willson,RichardC
中科院分区：
文献类型：
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作者：
Zhang,Xing;Potty,AjishSR;Jackson,GeorgeW;Stepanov,Victor;Tang,Andrew;Liu,Yamei;Kourentzi,Katerina;Strych,Ulrich;Fox,GeorgeE;Willson,RichardC
在以前的工作中，溶蛋白弧菌5S rRNA被证明可以稳定13-50个核苷酸的“客体”RNA序列，以便在大肠杆菌中表达。表达的嵌合RNA在E.大肠杆菌中的蛋白质不被掺入核糖体，对宿主细胞无明显影响。在这项工作中，我们将编码已知的识别蛋白质和有机染料的适体的序列插入到5S rRNA载体中，并表明适体功能在嵌合体中得以保留。表面等离子体共振竞争性结合试验表明，血管内皮生长因子（VEGF）适体/5S rRNA嵌合体在体外产生的转录径流可以与DNA适体竞争VEGF，这意味着生长因子的结合由VEGF“核糖体RNA适体”。另外，展示已知增加孔雀石绿色（MG）荧光的适体的5S rRNA嵌合体也增强MG荧光。密切相关的对照rRNA分子显示出两种活性。MG适体/5S rRNA嵌合体与原始MG适体一样，也增加了其他三苯基甲烷（TPM）染料如结晶紫、甲基紫和亮绿色的荧光，尽管不如MG有效。这些结果表明，当在稳定的5S rRNA载体的背景下表达适体时，适体的分子识别特性不会丧失。将适体包含在载体中可以促进大量RNA适体的产生，并且可以打开在体内筛选适体文库的方法。版权所有© 2009约翰威利父子有限公司。
In previous work,Vibrio proteolyticus5S rRNA was shown to stabilize 13–50 nucleotide “guest” RNA sequences for expression inEscherichia coli. The expressed chimeric RNAs accumulated to high levels inE. coliwithout being incorporated into ribosomes and without obvious effects on the host cells. In this work, we inserted sequences encoding known aptamers recognizing a protein and an organic dye into the 5S rRNA carrier and showed that aptamer function is preserved in the chimeras. A surface plasmon resonance competitive binding assay demonstrated that a vascular endothelial growth factor (VEGF) aptamer/5S rRNA chimera producedin vitroby transcriptional runoff could compete with a DNA aptamer for VEGF, implying binding of the growth factor by the VEGF “ribosomal RNA aptamer.” Separately, a 5S rRNA chimera displaying an aptamer known to increase the fluorescence of malachite green (MG) also enhanced MG fluorescence. Closely related control rRNA molecules showed neither activity. The MG aptamer/5S rRNA chimera, like the original MG aptamer, also increased the fluorescence of other triphenyl methane (TPM) dyes such as crystal violet, methyl violet, and brilliant green, although less effectively than with MG. These results indicate that the molecular recognition properties of aptamers are not lost when they are expressed in the context of a stable 5S rRNA carrier. Inclusion of the aptamer in a carrier may facilitate production of large quantities of RNA aptamers, and may open an approach to screening aptamer librariesin vivo. Copyright © 2009 John Wiley & Sons, Ltd.