Identification and characterization of nucleobase-modified aptamers by click-SELEX
Identification and characterization of nucleobase-modified aptamers by click-SELEX
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DOI：
10.1038/nprot.2018.023
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发表时间：
2018-05-01
期刊：
影响因子：
14.8
通讯作者：
Mayer, Guenter
中科院分区：
文献类型：
--
作者：
Pfeiffer, Franziska;Tolle, Fabian;Mayer, Guenter
适体是在体外选择以高亲和力和特异性识别其靶分子的单链寡核苷酸。由于它们由四种典型的核碱基组成，它们的化学多样性是有限的，这反过来又限制了可寻址的靶谱。将化学修饰引入核酸文库增加了DNA的相互作用能力，从而增加了靶谱。在这里，我们描述了一个协议来选择核碱基修饰的适体，通过使用点击化学（CuAAC）引入优选的化学修饰。使用点击化学修饰DNA文库使得能够引入广泛的可能功能，其可以根据靶分子和所需应用的要求进行定制。该方案产生具有扩展的相互作用性质的修饰的DNA适体，所述相互作用性质是用规范的核苷酸组不可及的。在合成含有市售的、炔修饰的尿苷（5-乙炔基-脱氧尿苷（EdU））而不是胸苷的起始文库后，通过CuAAC用所选的修饰将文库官能化。将如此修饰的DNA与靶分子孵育，并回收最佳结合序列。化学修饰在扩增过程中被去除。因此，该方案与常规扩增程序相容，并避免了与更广泛的核碱基修饰相关的酶促不相容性问题。单链生成后，将修饰重新引入富集文库中，然后可以对其进行随后的选择循环。方案中概述的每个选择周期的持续时间类似于1天。
Aptamers are single-stranded oligonucleotides that are in vitro-selected to recognize their target molecule with high affinity and specificity. As they consist of the four canonical nucleobases, their chemical diversity is limited, which in turn limits the addressable target spectrum. Introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of the DNA and thereby the target spectrum. Here, we describe a protocol to select nucleobase-modified aptamers by using click chemistry (CuAAC) to introduce the preferred chemical modification. The use of click chemistry to modify the DNA library enables the introduction of a wide range of possible functionalities, which can be customized to the requirements of the target molecule and the desired application. This protocol yields modified DNA aptamers with extended interaction properties that are not accessible with the canonical set of nucleotides. After synthesis of the starting library containing a commercially available, alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine, the library is functionalized with the modification of choice by CuAAC. The thus-modified DNA is incubated with the target molecule and the best binding sequences are recovered. The chemical modification is removed during the amplification process. Therefore, this protocol is compatible with conventional amplification procedures and avoids enzymatic incompatibility problems associated with more extensive nucleobase modifications. After single-strand generation, the modification is reintroduced into the enriched library, which can then be subjected to the subsequent selection cycle. The duration of each selection cycle as outlined in the protocol is similar to 1 d.