Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins
Streptavidin aptamers: Affinity tags for the study of RNAs and ribonucleoproteins
复制标题
DOI：
10.1017/s135583820100245x
复制
发表时间：
2001-04-01
期刊：
影响因子：
4.5
通讯作者：
Engelke, DR
中科院分区：
文献类型：
--
作者：
Srisawat, C;Engelke, DR
RNA亲和标签作为一种快速检测、固定和纯化的手段，对RNA和核糖核蛋白（RNPs）的研究非常有用。为了开发一种新的亲和标签，链亲和素结合RNA配体，称为“适体”，通过体外筛选从随机的RNA文库中鉴定出RNA/链亲和素复合物，并根据常见的序列将适配体分为两组，其中具有代表性的适配体具有足够低的解离常数，表明它们可能是有用的亲和工具。通过将链亲和素与生物素一起饱和，可以阻断适配体与链亲和素的结合，生物素可以用来解离RNA/链亲和素复合物。为了研究该适体作为亲和标签的实用性，我们将其中一个亲和性较高的适体插入到RNase P的大RNA亚基RPR1 RNA中。该适体标记的RNase P可以用市购的链亲和素琼脂糖特异性分离，并在温和条件下用生物素作为洗脱剂回收。适配体标签也被用来证明RNase P以单体形式存在，并且与RNase MRP（一种密切相关的核糖核蛋白酶）不紧密相关，这些结果表明链亲和素适配体是研究rna或RNPs的潜在强大工具。
RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection, Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools, Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme, These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.