Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation
Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation
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底物-模板稳定性、温度、磷酸盐位置和切口位点碱基对对非酶 DNA 连接的影响：定义用于优化碳二亚胺活化连接速率和产量的参数
DOI：
10.1101/821017
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
N. Hud
中科院分区：
文献类型：
--
作者：
C. Obianyor;G. Newnam;Bryce E. Clifton;M. Grover;N. Hud
非酶化学连接是用于产生合成DNA结构的重要工具，其用于广泛的应用。令人惊讶的是，对于相同的化学活化剂和相当的DNA结构，报道的化学连接产率范围为30%至95%。我们报告了一个系统的研究DNA连接使用一个定义良好的双分子测试系统和水溶性碳二亚胺（EDC）作为磷酸活化剂。我们的研究结果揭示了模板-底物稳定性和连接的化学步骤的速率之间的相互作用，这可能导致产量随着温度的升高而增加或减少。切口位点处的磷酸位置也表现出对连接速率和产率的强烈影响，对于特别有利的反应条件，3'磷酸在24小时后提供接近100%的产率，而在切口位点的5'位置上的磷酸的可比反应即使在48小时后也仅达到40%的连接。连接率也被证明是敏感的切口位点侧翼的碱基对的身份，与一些变化超过三倍。最后，在某些情况下，通过EDC修饰DNA底物会使长反应时间和重复添加EDC成为提高连接产率的无效策略。
Non-enzymatic, chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields range from 30% to 95% for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation using a well-defined bimolecular test system and water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results reveal interplay between template-substrate stability and the rates of the chemical steps of ligation, which can cause yields to increase or decrease with increasing temperature. Phosphate location at the nick site also exhibits a strong influence on ligation rates and yields, with a 3’ phosphate providing yields near 100% after 24 hours for particularly favourable reaction conditions, while comparable reactions with the phosphate on the 5’ position of the nick site only reach 40% ligation even after 48 hours. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with some varying by more than three-fold. Finally, DNA substrate modification by EDC can, in some cases, make long reaction times and repeated addition of EDC an ineffective strategy for increasing ligation yields.
DOI：
10.1002/anie.201506592
发表时间：
2015-11-23
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Jauker M;Griesser H;Richert C
通讯作者：
Richert C
影响因子：
16.6
作者：
Seeman NC
通讯作者：
Seeman NC