Single-round isolation of diverse RNA aptamers from a random sequence pool
Single-round isolation of diverse RNA aptamers from a random sequence pool
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DOI：
10.1093/biomethods/bpy004
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发表时间：
2018-05
影响因子：
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通讯作者：
Masahiko Imashimizu;Masaki Takahashi;Ryotaro Amano;Yoshikazu Nakamura
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文献类型：
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作者：
Masahiko Imashimizu;Masaki Takahashi;Ryotaro Amano;Yoshikazu Nakamura
摘要 适体是与靶分子具有特异性结合亲和力的寡核苷酸配体。通常，使用称为 SELEX 的技术从具有随机序列的 RNA 库中选择 RNA 适体，其中靶标结合的 RNA 分子被重复分离并呈指数扩增。尽管有几个优点，SELEX 在稀有靶标结合 RNA 分子的迭代扩增过程中经常产生不确定的结果。在这里，我们开发了一种无重复、无引物和无靶标固定的分离方法来生成 RNA 适体，该方法对实验噪声具有鲁棒性。独特的是，该方法侧重于通过 RNase 消化从 RNA 池中查找和去除非适体序列，留下靶标结合的适体分子，因此与适体类型无关。剩余的未消化的 RNA 序列数量如此之少，以至于必须与大量过量的已知序列混合以进行进一步操作，然后通过限制性消化去除该序列，然后进行高通量测序分析以鉴定适体。使用这种方法，我们独立地生成了多个针对 α-凝血酶和 TGFβ1 蛋白的 RNA 适体。该方法可能会生成数千个序列作为适体候选者，这可能使我们能够预测这些适体与输入RNA不同的共同平均序列或结构特性。
Abstract Aptamers are oligonucleotide ligands with specific binding affinity to target molecules. Generally, RNA aptamers are selected from an RNA pool with random sequences, using the technique termed SELEX, in which the target-binding RNA molecules are repeatedly isolated and exponentially amplified. Despite several advantages, SELEX often produces uncertain results during the iterative amplifications of the rare target-binding RNA molecules. Here, we develop a non-repeated, primer-less and target immobilization-free isolation method for generating RNA aptamers, which is robust to experimental noise. Uniquely, this method focuses on finding and removal of non-aptamer sequences from the RNA pool by RNase digestion leaving target-bound aptamer molecules, and thus is independent of aptamer types. The undigested RNA sequences remaining are so few in number that they must be mixed with a large excess of a known sequence for further manipulations and this sequence is then removed by restriction digestion followed by high-throughput sequencing analysis to identify aptamers. Using this method, we generated multiple RNA aptamers targeting α-thrombin and TGFβ1 proteins, independently. This method potentially generates thousands of sequences as aptamer candidates, which may enable us to predict a common average sequence or structural property of these aptamers that is different from input RNA.