Edeleva Evgeniia, Salditt Annalena, Stamp Julian, Schwintek Philipp, Boekhoven Job, Braun Dieter
Systems Biophysics , Physics Department , Ludwig-Maximilians-Universität München , Amalienstraße 54 , 80799 München , Germany . Email:
Chemistry Department and Institute for Advanced Study , Technical University of Munich , Lichtenbergstraße 4 , 80895 Garching , Germany.
Chem Sci. 2019 Apr 30;10(22):5807-5814. doi: 10.1039/c9sc00770a. eCollection 2019 Jun 14.
Continuous enzyme-free replication of oligonucleotides is central for open-ended evolution experiments that mimic the origin of life. Here, we studied a reaction system, whereby two 24mer DNA templates cross-catalyzed each other's synthesis from four 12mer DNA fragments, two of which were activated with the condensing agent 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). We circumvented the problem of product inhibition by melting the stable product duplexes for their reuse as templates in the following ligation step. The system reproduced itself through ligation/melting cycles and survived exponential dilution. We quantified EDC-induced side reactions in a detailed kinetic model. The model allowed us to analyze the effects of various reaction rates on the system's kinetics and confirmed maximal replication under the chosen conditions. The presented system enables us to study nonenzymatic open-ended evolution experiments starting from diverse sequence pools.
寡核苷酸的连续无酶复制对于模拟生命起源的开放式进化实验至关重要。在此，我们研究了一个反应体系，其中两条24聚体DNA模板由四个12聚体DNA片段相互交叉催化合成，其中两个片段用缩合剂1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）活化。我们通过熔解稳定的产物双链体来避免产物抑制问题，以便在后续连接步骤中将其作为模板重新使用。该体系通过连接/熔解循环自我复制，并在指数稀释下存活。我们在一个详细的动力学模型中对EDC诱导的副反应进行了量化。该模型使我们能够分析各种反应速率对体系动力学的影响，并证实了在所选条件下的最大复制。所展示的体系使我们能够从不同的序列库开始研究非酶促开放式进化实验。
