1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA
1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA
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DOI：
10.1002/cbic.201700185
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发表时间：
2017-08
期刊：
影响因子：
3.2
通讯作者：
Krisana Peewasan;H. Wagenknecht
中科院分区：
文献类型：
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作者：
Krisana Peewasan;H. Wagenknecht
为了建立Diels-Alder反应，合成了一种1,2,4 -三嗪修饰的2 ' -脱氧尿苷三磷酸。生物正交反应的1,2,4 -三嗪基团通过柔性烷基连接连接在2 ' -脱氧尿嘧啶的5 -位置，以促进其被DNA聚合酶接受。四种DNA聚合酶的筛选表明，通过KOD XL或Vent聚合酶，使用dATP、dGTP、dCTP和修饰的2 '‐脱氧尿苷三磷酸的混合物，成功地扩展了引物。在引物延伸的条件下，三嗪部分是稳定的，在室温下用BCN修饰的罗丹明标记可以证明这一点，收率高达82%。当修饰位点被三个碱基对分开时，两个或三个修饰碱基可以被纳入定量产率。这些结果确定了1,2,4‐三氮烯基团是DNA中的生物正交反应基团，从而取代了有问题的1,2,4,5‐四氮，用于Diels-Alder反应的合成后标记。
In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.