School of Chemistry and Chemical Engineering, Institute of Molecular Medicine Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, P.R., China.
School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules and National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai, 200240, P.R. China.
Chempluschem. 2024 Jun;89(6):e202300781. doi: 10.1002/cplu.202300781. Epub 2024 Mar 1.
Efficient biocatalytic cascade reactions play a crucial role in guiding intricate, specific and selective intracellular transformation processes. However, the catalytic activity of the enzyme cascade reaction in bulk solution was greatly impacted by the spatial morphology and inter-enzyme distance. The programmability and addressability nature of framework nucleic acid (FNA) allows to be used as scaffold for immobilization and to direct the spatial arrangement of enzyme cascade molecules. Here, we used tetrahedral DNA framework (TDF) as nanorulers to assemble two enzymes for constructing a double-enzyme complex, which significantly enhance the catalytic efficiency of sarcosine oxidase (SOx)/horseradish peroxidase (HRP) cascade system. We synthesized four types of TDF nanorulers capable of programming the lateral distance between enzymes from 5.67 nm to 12.33 nm. Enzymes were chemical modified by ssDNA while preserving most catalytic activity. Polyacrylamide gel electrophoresis (PAGE), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to verify the formation of double-enzyme complex. Four types of double-enzyme complexes with different enzyme distance were constructed, in which TDF(SOx+HRP) exhibited the highest relative enzyme cascade catalytic activity, ~3.11-fold of free-state enzyme. Importantly, all the double-enzyme complexes demonstrate a substantial improvement in enzyme cascade catalytic activity compared to free enzymes.
有效的生物催化级联反应在指导复杂、特异和选择性的细胞内转化过程中起着至关重要的作用。然而，在体相溶液中，酶级联反应的催化活性受到空间形态和酶间距离的极大影响。框架核酸（FNA）的可编程性和寻址性可以作为固定化的支架，并指导酶级联分子的空间排列。在这里，我们使用四面体 DNA 框架（TDF）作为纳米标尺来组装两种酶，构建双酶复合物，从而显著提高了肌氨酸氧化酶（SOx）/辣根过氧化物酶（HRP）级联系统的催化效率。我们合成了四种 TDF 纳米标尺，能够将酶之间的侧向距离编程为 5.67nm 至 12.33nm。酶通过 ssDNA 进行化学修饰，同时保留了大部分催化活性。我们使用聚丙烯酰胺凝胶电泳（PAGE）、透射电子显微镜（TEM）和原子力显微镜（AFM）来验证双酶复合物的形成。构建了四种具有不同酶间距的双酶复合物，其中 TDF（SOx+HRP）表现出最高的相对酶级联催化活性，约为游离态酶的 3.11 倍。重要的是，与游离酶相比，所有的双酶复合物都显著提高了酶级联催化活性。
