Chemical strategies that augment genetic polymers with amino acid residues that are overrepresented on the paratope surface of an antibody offer a promising route for enhancing the binding properties of nucleic acid aptamers. Here, we describe the chemical synthesis of α-L-threofuranosyl cytidine nucleoside triphosphate (tCTP) carrying either a benzyl or phenylpropyl side chain at the pyrimidine C-5 position. Polymerase recognition studies indicate that both substrates are readily incorporated into a full-length α-L-threofuranosyl nucleic acid (TNA) product by extension of a DNA primer-template duplex with an engineered TNA polymerase. Similar primer extension reactions performed using nucleoside triphosphate mixtures containing both C-5 modified tCTP and C-5 modified tUTP substrates enable the production of doubly modified TNA strands for a panel of 20 chemotype combinations. Kinetic measurements reveal faster on-rates (k_on) and tighter binding affinity constants (K_d) for engineered versions of TNA aptamers carrying chemotypes at both pyrimidine positions as compared to their singly modified counterparts. These findings expand the chemical space of evolvable non-natural genetic polymers by offering a path for improving the quality of biologically stable TNA aptamers for future clinical applications.

中文翻译：

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增加苏糖核酸的功能密度

用抗体互补位表面上过量表达的氨基酸残基增强遗传聚合物的化学策略为增强核酸适体的结合特性提供了一条有前途的途径。在这里，我们描述了在嘧啶 C-5 位带有苄基或苯丙基侧链的α- L-苏呋喃糖基胞苷三磷酸 (tCTP)的化学合成。聚合酶识别研究表明，通过用工程化 TNA 聚合酶延伸 DNA 引物-模板双链体，两种底物很容易掺入全长 α- L-苏呋喃糖核酸 (TNA) 产物中。使用含有 C-5 修饰的 tCTP 和 C-5 修饰的 tUTP 底物的核苷三磷酸混合物进行类似的引物延伸反应，能够为一组 20 种化学型组合产生双重修饰的 TNA 链。动力学测量表明，与单独修饰的对应物相比，在两个嘧啶位置携带化学型的 TNA 适配体的工程版本具有更快的结合速率 ( k _on ) 和更紧密的结合亲和力常数 ( K _{d )。}这些发现为提高未来临床应用中生物稳定的 TNA 适体的质量提供了一条途径，扩大了可进化非天然遗传聚合物的化学空间。