A general strategy for direct, enzyme-catalyzed conjugation of functional compounds to DNA.
A general strategy for direct, enzyme-catalyzed conjugation of functional compounds to DNA.
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DOI：
10.1093/nar/gky184
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发表时间：
2018-06-20
影响因子：
14.9
通讯作者：
Neely RK
中科院分区：
文献类型：
--
作者：
Deen J;Wang S;Van Snick S;Leen V;Janssen K;Hofkens J;Neely RK
甲基转移酶可以应用于以特殊的特异性和效率对大DNA分子上预先确定的位点进行一系列修饰。然而，迄今为止，以这种方式进行的修饰数量有限，因为需要复杂的化学合成来产生具有特定功能的辅因子类似物，例如荧光团。在这里，我们描述了一种将一系列功能化合物（七种荧光染料、生物素和聚乙二醇）直接转移到DNA双链上的方法。我们的方法使用功能辅助因子类似物，其最后的制备步骤与DNA修饰反应一起在单个锅中进行，不需要纯化。我们表明，与两步标记方法相比，这些混合物中的荧光团共轭效率显着提高。我们的实验突出了甲基转移酶的显著延展性和选择性。对荧光团分布进行高分辨率定位的进一步分析表明，甲基转移酶的靶位点主要在其回文位点的单链上进行标记，并且存在小且随机分布的非位点标记概率。
The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex. Our approach uses a functional cofactor analogue, whose final preparative step is performed alongiside the DNA modification reaction in a single pot, with no purification needed. We show that fluorophore conjugation efficiency in these mixtures is significantly improved compared to two-step labeling approaches. Our experiments highlight the remarkable malleability and selectivity of the methyltransferases tested. Additional analysis using high resolution localization of the fluorophore distribution indicates that target sites for the methyltransferase are predominantly labeled on a single strand of their palindromic site and that a small and randomly-distributed probability of off-site labeling exists.
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DOI：
10.1002/anie.201608625
发表时间：
2017-05-02
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Deen J;Vranken C;Leen V;Neely RK;Janssen KPF;Hofkens J
通讯作者：
Hofkens J
影响因子：
17.1
作者：
Deen, Jochem;Sempels, Wouter;De Dier, Raf;Vermant, Jan;Dedecker, Peter;Hofkens, Johan;Neely, Robert K.
通讯作者：
Neely, Robert K.
影响因子：
8.4
作者：
Neely, Robert K.;Dedecker, Peter;Hofkens, Johan
通讯作者：
Hofkens, Johan
影响因子：
14.9
作者：
Lukinavicius G;Lapinaite A;Urbanaviciute G;Gerasimaite R;Klimasauskas S
通讯作者：
Klimasauskas S
影响因子：
14.8
作者：
Dalhoff, C;Lukinavicius, G;Weinhold, E
通讯作者：
Weinhold, E