Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
J Am Chem Soc. 2010 Mar 31;132(12):4141-51. doi: 10.1021/ja908035g.
Six new 5-position modified dUTP derivatives connected by a unique amide linkage were synthesized and tested for compatibility with the enzymatic steps of in vitro selection. Six commercially available DNA polymerases were tested for their ability to efficiently incorporate each of these dUTP derivatives during PCR. It was not possible to perform PCR under standard conditions using any of the modified dUTP derivatives studied. In contrast, primer extension reactions of random templates, as well as defined sequence templates, were successful. KOD XL and D. Vent DNA polymerases were found to be the most efficient at synthesizing full-length primer extension product, with all of the dUTP derivatives tested giving yields similar to those obtained with TTP. Several of these modified dUTPs were then used in an in vitro selection experiment comparing the use of modified dUTP derivatives with TTP for selecting aptamers to a protein target (necrosis factor receptor superfamily member 9, TNFRSF9) that had previously been found to be refractory to in vitro selection using DNA. Remarkably, selections employing modified DNA libraries resulted in the first successful isolation of DNA aptamers able to bind TNFRSF9 with high affinity.
合成了六个新的 5 位修饰的 dUTP 衍生物，通过独特的酰胺键连接，用于测试与体外选择的酶学步骤的兼容性。测试了六种市售的 DNA 聚合酶，以确定它们在 PCR 过程中有效掺入每种 dUTP 衍生物的能力。使用研究的任何修饰的 dUTP 衍生物都不可能在标准条件下进行 PCR。相比之下，随机模板和定义序列模板的引物延伸反应是成功的。发现 KOD XL 和 D. Vent DNA 聚合酶在合成全长引物延伸产物方面效率最高，所有测试的 dUTP 衍生物的产率与 TTP 获得的产率相似。然后在体外选择实验中使用了其中几种修饰的 dUTP，比较了使用修饰的 dUTP 衍生物与 TTP 选择对先前发现对使用 DNA 进行体外选择有抗性的蛋白质靶标（坏死因子受体超家族成员 9，TNFRSF9）的适体。值得注意的是，修饰的 DNA 文库的选择导致首次成功分离到能够与 TNFRSF9 高亲和力结合的 DNA 适体。
