用指数富集方法鉴定抗志贺毒素RNA适体的配体选择进化。

IF 1.3 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Nucleic Acids Pub Date : 2014-01-01 Epub Date: 2014-04-15 DOI:10.1155/2014/214929
Sreerupa Challa, Saul Tzipori, Abhineet Sheoran
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引用次数: 14

摘要

感染产生志贺毒素(Stx-)的大肠杆菌可导致危及生命的溶血性尿毒症综合征(HUS),这是儿童急性肾衰竭的主要原因。在Stx1和Stx2两种抗原性不同的毒素中,Stx2与溶血性毒血症的发生联系更为紧密。在本研究中,通过指数富集的选择性进化配体(SELEX)来鉴定抗Stx1和Stx2的RNA适体。经过5轮筛选,使用含有56个随机核苷酸(N56)的RNA适体文库,获得了针对Stx2而非Stx1的显著富集的适体库。对核酸适配体序列的分析表明,有6个独特的RNA适配体(mA/pC、mB/pA、mC、mD、pB和pD)在过滤结合实验中识别Stx2。这些适体都没有结合Stx1。流式细胞术检测显示,适配体mA/pC、mB/pA、mC和mD,但不包括pB和pD,部分阻断了Alexa 488标记的Stx2与HeLa细胞的结合。然而,没有一种适体能中和stx2介导的HeLa细胞的细胞毒性和死亡。
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Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins.

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.

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来源期刊
Journal of Nucleic Acids
Journal of Nucleic Acids BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
3.10
自引率
21.70%
发文量
5
审稿时长
12 weeks
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