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{"title":"Determining Functional Aptamer-Protein Interaction by Biolayer Interferometry","authors":"Xinhui Lou, Martin Egli, Xianbin Yang","doi":"10.1002/cpnc.18","DOIUrl":null,"url":null,"abstract":"<p>Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol). We also report on the protocol for the affinity screening of a panel of anti-thrombin RNA aptamers with a single phosphorodithioate (PS2) modification, whereby the position of the modification along the RNA backbone is varied systematically (Support Protocol). The PS2 modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphodiester backbone linkage. The PS2-modified RNAs are nuclease resistant and several in vitro and in vivo assays have demonstrated their biological activity. For example, combining the PS2 with the 2′-OMe modification affords increased loading of modified small interfering RNA (siRNA) duplexes into the RNA-induced silencing complex (RISC) as well as enhanced gene-silencing antitumor activity. © 2016 by John Wiley & Sons, Inc.</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":"67 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.18","citationCount":"20","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Nucleic Acid Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpnc.18","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Chemistry","Score":null,"Total":0}
引用次数: 20
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Abstract
Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol). We also report on the protocol for the affinity screening of a panel of anti-thrombin RNA aptamers with a single phosphorodithioate (PS2) modification, whereby the position of the modification along the RNA backbone is varied systematically (Support Protocol). The PS2 modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphodiester backbone linkage. The PS2-modified RNAs are nuclease resistant and several in vitro and in vivo assays have demonstrated their biological activity. For example, combining the PS2 with the 2′-OMe modification affords increased loading of modified small interfering RNA (siRNA) duplexes into the RNA-induced silencing complex (RISC) as well as enhanced gene-silencing antitumor activity. © 2016 by John Wiley & Sons, Inc.
生物层干涉法测定功能适配体-蛋白质相互作用
被称为适体的短单链核酸作为一种高亲和力和特异性的识别分子被广泛探索,用于结合广泛的靶分子,特别是蛋白质靶标。在生物层干涉法(BLI)中,一种简单的浸读法(Dip-and-Read)用于研究适体与其靶蛋白之间的相互作用,该方法将包裹适体的生物传感器浸入微孔板中。在这里,我们描述的方案分析之间的相互作用的抗凝血酶RNA适配体与凝血酶(基本方案)。我们还报道了一组具有单一硫代磷酸酯(PS2)修饰的抗凝血酶RNA适体的亲和力筛选方案,其中沿RNA主干的修饰位置是系统地变化的(支持方案)。PS2修饰使用两个硫原子取代核苷酸间磷酸二酯主链上的两个非桥接氧原子。ps2修饰的rna具有核酸酶抗性,并且一些体外和体内实验已经证明了它们的生物活性。例如,将PS2与2 ' -OMe修饰相结合,可以增加修饰后的小干扰RNA (siRNA)双链加载到RNA诱导的沉默复合体(RISC)中,并增强基因沉默抗肿瘤活性。©2016 by John Wiley &儿子,Inc。
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