Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins.
Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins.
复制标题
通过指数富集选择性进化配体来识别抗志贺毒素的 RNA 适体。
DOI：
10.1155/2014/214929
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发表时间：
2014
影响因子：
2.3
通讯作者：
Sheoran A
中科院分区：
文献类型：
--
作者：
Challa S;Tzipori S;Sheoran A
感染产志贺毒素(STX-)的大肠杆菌会导致危及生命的溶血性尿毒症综合征(HUS)，这是导致儿童急性肾功能衰竭的主要原因。在两种抗原性不同的毒素STX1和Stx2中，Stx2与HUS的发展更紧密地联系在一起。在本研究中，通过指数富集法(SELEX)选择性进化配体，试图确定针对STX1和STX2的RNA适配子。经过5轮筛选，利用包含56个随机核苷酸(N56)的RNA适配子文库，对Stx2获得了显著的适配子库，但对STX1没有得到显著的丰富。对单个适配子序列的分析表明，在过滤器结合试验中，六个独特的RNA适配子(mA/PC、MB/PA、MC、Md、PB和Pd)能够识别Stx2。这些适配子都不能与STX1结合。在流式细胞术分析中，适体Ma/PC、MB/PA、MC和MD，而不是PB和PD，部分阻断了Alexa 488标记的Stx2与HeLa细胞的结合。然而，这些适配子都不能中和Stx2介导的HeLa细胞的细胞毒性和死亡。
Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells.