Zhou Shaohong, Ju Xiuqin, Chen Hui, Zhang Yanwen, Zheng Jing, Wang Kemin, Yang Xiaohai, Liu Jianbo
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha, 410082, P.R. China.
Angew Chem Int Ed Engl. 2025 Jul 28;64(31):e202505316. doi: 10.1002/anie.202505316. Epub 2025 Jun 2.
Precise control of enzymatic cascade pathways is essential for deepening our understanding of metabolic processes and designing synthetic biochemical systems. In this study, we present a novel approach to modulate these cascades using light-responsive DNA droplets. Y-motif sequences with azobenzene-modified sticky ends (SE) were designed to form DNA droplets via liquid-liquid phase separation. These droplets enable selective sequestration of enzymes within compartments, allowing dynamic regulation of enzymatic cascades in response to ultraviolet (UV) and visible (vis) light stimuli. As a model system, we employed an incompatible enzymatic cascade, involving glucose oxidase (GOx), horseradish peroxidase (HRP), and catalase (CAT). Under visible light, the DNA droplets co-localize enzymes within the compartments, effectively regulating metabolic flux without altering the total enzyme concentration. Upon UV exposure, the droplets disassemble, releasing the sequestered enzymes and altering cascade dynamics. The versatility of this approach was further demonstrated using an alternative enzymatic cascade system comprising galactose oxidase (GAO), myeloperoxidase (MPO), and CAT. This system demonstrates that light-responsive DNA droplets are a powerful tool for regulating enzymatic pathways, with potential applications in synthetic biology, microreactor design, and bioengineering.
精确控制酶促级联反应途径对于深化我们对代谢过程的理解以及设计合成生化系统至关重要。在本研究中，我们提出了一种利用光响应性DNA液滴来调节这些级联反应的新方法。设计了具有偶氮苯修饰粘性末端（SE）的Y基序序列，通过液-液相分离形成DNA液滴。这些液滴能够在隔室内选择性地隔离酶，从而响应紫外线（UV）和可见光（vis）刺激对酶促级联反应进行动态调节。作为一个模型系统，我们采用了一个不相容的酶促级联反应，涉及葡萄糖氧化酶（GOx）、辣根过氧化物酶（HRP）和过氧化氢酶（CAT）。在可见光下，DNA液滴将酶共定位在隔室内，在不改变总酶浓度的情况下有效调节代谢通量。暴露于紫外线后，液滴解体，释放被隔离的酶并改变级联反应动力学。使用包含半乳糖氧化酶（GAO）、髓过氧化物酶（MPO）和CAT的另一种酶促级联反应系统进一步证明了该方法的通用性。该系统表明，光响应性DNA液滴是调节酶促途径的有力工具，在合成生物学、微反应器设计和生物工程中具有潜在应用。
