Profiling the selectivity of DNA ligases in an array format with mass spectrometry.
Profiling the selectivity of DNA ligases in an array format with mass spectrometry.
复制标题
DOI：
10.1093/nar/gkp827
复制
发表时间：
2010-01
影响因子：
14.9
通讯作者：
Mrksich M
中科院分区：
文献类型：
--
作者：
Kim J;Mrksich M
本文介绍了一种方法的整体分析的底物特异性的DNA连接酶，并举例说明使用Taq和T4 DNA连接酶。该方法结合了寡核苷酸阵列，这提供了高通量和多重测定的好处，与质谱允许连接酶活性的无标记测定。通过将三元生物素标记的DNA底物固定到呈现链霉亲和素蛋白层的自组装单层来制备阵列。该阵列表示在切口位点的3′侧具有所有可能的匹配和错配碱基对的复合物，并且还包括在该位点的许多缺失和插入。用连接酶和三磷酸腺苷或三磷酸核苷类似物处理阵列，然后通过基质辅助激光解吸电离质谱法进行分析，以确定5′-探针链的腺苷酸化和两条探针链的连接的产率。由此产生的活性曲线揭示了连接酶特异性的基础，也指出了使用ATP类似物来提高特异性的策略。这项工作介绍了一种方法，可以适用于分析广泛的酶上的核酸底物。
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.
登录
查看更多内容
影响因子：
14.9
作者：
Bhagwat, AS;Sanderson, RJ;Lindahl, T
通讯作者：
Lindahl, T
影响因子：
9.3
作者：
Bonk, T;Humeny, A;Becker, CM
通讯作者：
Becker, CM
影响因子：
14.9
作者：
Coolen, Marcel W;Statham, Aaron L;Gardiner-Garden, Margaret;Clark, Susan J
通讯作者：
Clark, Susan J
影响因子：
46.9
作者：
Min, DH;Tang, WJ;Mrksich, M
通讯作者：
Mrksich, M
影响因子：
3.9
作者：
Houseman, BT;Gawalt, ES;Mrksich, M
通讯作者：
Mrksich, M