Clustering of catalytic nanocompartments for enhancing an extracellular non-native cascade reaction

Abstract

Compartmentalization is fundamental in nature, where the spatial segregation of biochemical reactions within and between cells ensures optimal conditions for the regulation of cascade reactions. While the distance between compartments or their interaction are essential parameters supporting the efficiency of bio-reactions, so far they have not been exploited to regulate cascade reactions between bioinspired catalytic nanocompartments. Here, we generate individual catalytic nanocompartments (CNCs) by encapsulating within polymersomes or attaching to their surface enzymes involved in a cascade reaction and then, tether the polymersomes together into clusters. By conjugating complementary DNA strands to the polymersomes' surface, DNA hybridization drove the clusterization process of enzyme-loaded polymersomes and controlled the distance between the respective catalytic nanocompartments. Owing to the close proximity of CNCs within clusters and the overall stability of the cluster architecture, the cascade reaction between spatially segregated enzymes was significantly more efficient than when the catalytic nanocompartments were not linked together by DNA duplexes. Additionally, residual DNA single strands that were not engaged in clustering, allowed for an interaction of the clusters with the cell surface as evidenced by A549 cells, where clusters decorating the surface endowed the cells with a non-native enzymatic cascade. The self-organization into clusters of catalytic nanocompartments confining different enzymes of a cascade reaction allows for a distance control of the reaction spaces which opens new avenues for highly efficient applications in domains such as catalysis or nanomedicine.

Fig. 1. Concepts of a GOX–LPO cascade between two clustered CNCs, tethered via complementary ssDNA, in order to facilitate the diffusion of H₂O₂ and thus improve the overall reaction efficiency. Similarly, an AMG–GOX–LPO cascade achieves an improved diffusion of the glucose derived from amylose, and the enzyme on the surface allows the access to bulky substrates that would otherwise be out of reach for encapsulated enzymes.

Fig. 2. (A) TEM micrograph of GOX-CNC. (B) TEM micrograph of LPO-CNC. (C) FCS autocorrelation curves of free Atto-488 (black), Atto-488–GOX (red) and Atto-488–GOX-loaded CNCs (blue). Dots: raw data. Line: fitted model. (D) FCS autocorrelation curves of free DyLight-633 (black), DyLight-633–LPO (red) and DyLight-633–LPO-loaded CNCs (blue).

Fig. 3. (A) Activity of LPO-CNC with melittin (blue), CNCs without melittin (red) and substrates alone (black, hidden behind red). (B) Activity of GOX-CNC with melittin (blue), CNCs without melittin (red) and substrates alone (black), using LPO as reporter enzyme. (C) Activity of AMG(GOX)-CNC with melittin (blue), without melittin (red) and substrates alone (black). Error bands represent ±SD, n = 3 replicates.

Fig. 4. (A) Time course of cluster growth starting form unclustered CNCs by DLS. Blue: LPO–GOX-CNC clusters; red: AMG(GOX)–LPO-CNC clusters. (B) FCS autocorrelation curves of unclustered GOX–ATTO488 – loaded CNCs (red) and unclustered LPO–DyLight 633 – loaded CNCs (green), and FCCS curves of clustered (blue) and unclustered (black) CNCs. Dots: raw data. Line: fitted model. (C) TEM micrographs of CNC clusters at different magnifications: left pannel, scale bar = 200 nm, right pannel, scale bar = 100 nm.

Fig. 5. (A) Concept of a clustered GOX–LPO-CNC cascade and the enzymatic activity of CNC clusters (blue), unclustered CNCs (red) and Amplex Red autoxidation (black). (B) Concept of a clustered AMX(GOX)–LPO-CNC cascade and the enzymatic activity of CNC clusters (blue), unclustered CNCs (red), GOX–LPO-CNC with AMG in solution (magenta) and Amplex Red autoxidation (black). Error bands given as ±SD, n = 3.

Fig. 6. (A) Localization of clustered GOX-CNCs (green) on A549 cells. (B) Same area with localization of LPO-CNCs (red). (C) Merged channels revealing colocalization of clustered GOX- and LPO CNCs (white). (D) Transmission channel. (E) Overlay including the transmission channel. Insets show higher magnifications of the areas boxed in white. The blurred appearance of the brightfield image indicates that the clusters are on the cell surface. Scalebars, 10 μm.

Fig. 7. (A) Activity of GOX–LPO clusters on the surface of cells (blue) and AR non-specific oxidation by cells (black). (B) Activity of AMG(GOX)–LPO clusters on the surface of cells (blue), and AR non-specific oxidation by cells (black). Error bands given as ±SD, n = 3.