Truncation and Mutation of a Transferrin Receptor Aptamer Enhances Binding Affinity
Truncation and Mutation of a Transferrin Receptor Aptamer Enhances Binding Affinity
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DOI：
10.1089/nat.2015.0585
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发表时间：
2016-12-01
影响因子：
4
通讯作者：
Shigdar, Sarah
中科院分区：
文献类型：
--
作者：
Macdonald, Joanna;Houghton, Patrick;Shigdar, Sarah
适体在许多应用中证明了它们的实用性。然而，为了便于功能化，它们的结构需要简化。因此，我们试图利用较长亲本序列的预测二维结构的合理工程，将转铁蛋白受体特异性的50个核苷酸的适体截断到其最小的功能单元。此外，将突变引入结合环以确定其对适体选择性的影响。这些碱基突变增强了适体的结合亲和力，同时保留了其特异性。平衡解离常数（Kd）降低了六倍后，取代所有四个碱基在结合区。此外，这些适体以与转铁蛋白受体抗体相似的方式有效地内化到转铁蛋白受体阳性细胞中，并与该抗体共定位。该研究表明，该适体的最小功能单元为14个核苷酸。这种小尺寸将有利于未来的应用，例如药物输送或其他治疗方式的功能化。
Aptamers are proving their utility in a number of applications. However, to be easily functionalized, their structure needs to be simplified. Therefore, we sought to truncate a 50-nucleotide aptamer specific to the transferrin receptor to its smallest functional unit using rational engineering of the predicted two-dimensional structure of the longer parent sequence. In addition, mutations were introduced into the binding loop to determine their effect on the selectivity of the aptamers. These base mutations enhanced the binding affinity of the aptamer, while retaining its specificity. The equilibrium dissociation constant (Kd) was reduced sixfold following the substitution of all four bases in the binding region. In addition, these aptamers were efficiently internalized into transferrin receptor-positive cells in a similar manner to the transferrin receptor antibody and demonstrated colocalization with this antibody. This study has shown that the smallest functional unit of this aptamer was 14 nucleotides. This small size will be advantageous for future applications, such as drug delivery or functionalization of other therapeutic modalities.