Department of Chemistry, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA.
Nucleic Acids Res. 2010 Jan;38(1):e2. doi: 10.1093/nar/gkp827. Epub 2009 Oct 23.
This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3' side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5'-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates.
本文描述了一种用于对 DNA 连接酶的底物特异性进行全局分析的方法，并以 Taq 和 T4 DNA 连接酶为例进行了说明。该方法结合了寡核苷酸阵列，其具有高通量和多重分析的优势，以及质谱，以允许对连接酶活性进行无标记分析。阵列通过将三碱基生物素标记的 DNA 底物固定在自组装单层上而制备，该自组装单层呈现一层链霉亲和素蛋白。该阵列代表了在切口位点的 3' 侧具有所有可能的匹配和不匹配碱基对的复合物，并且还包括在该位点的一些缺失和插入。用连接酶和三磷酸腺苷或三磷酸核苷酸类似物处理阵列，然后通过基质辅助激光解吸电离质谱分析来确定 5' -探针链的腺苷酸化和两个探针链的连接的产率。所得的活性谱揭示了连接酶特异性的基础，并指出了使用 ATP 类似物来提高特异性的策略。这项工作介绍了一种可以用于对作用于核酸底物的广泛范围的酶进行分析的方法。
