Investigations toward the selection of fully-modified 4′-thioRNA aptamers: Optimization of in vitro transcription steps in the presence of 4′-thioNTPs
Investigations toward the selection of fully-modified 4′-thioRNA aptamers: Optimization of in vitro transcription steps in the presence of 4′-thioNTPs
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DOI：
10.1016/j.bmc.2008.09.048
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发表时间：
2008-11-01
影响因子：
3.5
通讯作者：
Matsuda, Akira
中科院分区：
文献类型：
--
作者：
Minakawa, Noriaki;Sanji, Mioko;Matsuda, Akira
在此，我们描述了一种使用SELEX方案分离抗人α-凝血酶的完全修饰的4‘-硫代RNA适配子的方法。为了分离出所需的适配子，在4种4‘-硫代核苷三磷酸(4’-thioNTPs)存在下进行体外转录实验，以获得完全修饰的4‘-thioRNA。首先比较了4‘-硫代NTPs和5’-(α-硫代)三磷酸核苷(α-SNTPs)的转录效率，发现它们的转录效率不如aSNTPs，特别是当GTP和/或ATP被4‘-硫代GTP和/或4’-硫代ATP取代时。进一步提高其效率的尝试，包括使用突变的RNA聚合酶而不是野生型，各种添加剂和4‘-硫代NTP浓度都没有成功。因此，转录是在4‘-硫代NTPs存在的情况下进行的，并在适当浓度下与天然GTP和ATP一起进行。虽然这一尝试提供了高度修饰的4‘-thioRNA，但不是完全修饰的4’-thioRNA，但我们最终利用优化的转录条件通过SELEX成功地分离出了完全修饰的4‘-thioRNA适配子，随后对得到的适配子进行了后修饰。(C)2008爱思唯尔有限公司。保留所有权利。
We describe herein a method for isolating fully-modified 4 '-thioRNA aptamers against human alpha-thrombin using the SELEX protocol. In order to isolate the desired aptamers, in vitro transcription was examined in the presence of four kinds of 4 '-thioribonucleoside triphosphates (4 '-thioNTPs) in an effort to afford the fully-modified 4 '-thioRNA. The transcription efficiency of the 4 '-thioNTPs was first compared with that of the nucleoside 5 '-(alpha-thio)triphosphates (alpha SNTPs) and found to be less effective than that of the aSNTPs, especially when GTP and/or ATP were substituted for 4 '-thioGTP and/or 4 '-thioATP. Further attempts to improve its efficiency, including the use of a mutant RNA polymerase instead of the wild type, various additives, and 4 '-thioNTP concentrations were unsuccessful. Accordingly, the transcription was performed in the presence of 4 '-thioNTPs together with the natural GTP and ATP at the appropriate concentrations. Although this attempt furnished a highly-modified 4 '-thioRNA, but not a fully-modified 4 '-thioRNA, we eventually succeeded in isolating the fully-modified 4 '-thioRNA aptamers by SELEX using optimized transcription conditions, followed by post-modification of the resulting aptamers. (C) 2008 Elsevier Ltd. All rights reserved.