Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia.
Novosibirsk State University, Novosibirsk, Russia.
Dokl Biochem Biophys. 2024 Oct;518(1):376-381. doi: 10.1134/S1607672924600416. Epub 2024 Aug 28.
Click ligation is a technology of joining DNA fragments based on azide-alkyne cycloaddition. In the most common variant, click ligation introduces a 4-methyl-1,2,3-triazole (trz) group instead of the phosphodiester bond between two adjacent nucleosides. While this linkage is believed to be biocompatible, little is known about the possibility of its recognition by DNA repair systems or its potential for DNA polymerase stalling and miscoding. Here we report that trz linkage is resistant to several human and bacterial endonucleases involved in DNA repair. At the same time, it strongly blocks some DNA polymerases (Pfu, DNA polymerase β) while allowing bypass by others (phage RB69 polymerase, Klenow fragment). All polymerases, except for DNA polymerase β, showed high frequency of misinsertion at the trz site, incorporating dAMP instead of the next complementary nucleotide. Thus, click ligation can be expected to produce a large amount of errors if used in custom gene synthesis.
点击连接是一种基于叠氮-炔环加成反应连接 DNA 片段的技术。在最常见的变体中，点击连接引入一个 4-甲基-1,2,3-三唑（trz）基团，而不是两个相邻核苷酸之间的磷酸二酯键。虽然这种键合被认为是生物兼容的，但关于其被 DNA 修复系统识别的可能性或其导致 DNA 聚合酶停滞和错配的潜力知之甚少。在这里，我们报告 trz 键合能够抵抗几种参与 DNA 修复的人类和细菌内切酶。同时，它强烈阻止了一些 DNA 聚合酶（Pfu、DNA 聚合酶β），而允许其他聚合酶（噬菌体 RB69 聚合酶、Klenow 片段）绕过它。除了 DNA 聚合酶β之外，所有聚合酶在 trz 位点的插入错误频率都很高，会掺入 dAMP 而不是下一个互补核苷酸。因此，如果在定制基因合成中使用点击连接，预计会产生大量错误。
