DNA-assembled nanofactories represent a cutting-edge approach in metabolic therapy, leveraging the programmability of DNA nanostructures to create spatially organized enzymatic systems. These nanofactories enable precise co-localization of multiple enzymes, mimicking natural metabolic pathways to enhance reaction efficiency. By arranging enzymes in close proximity, substrate channeling is facilitated, reducing diffusion limitations and improving catalytic turnover. This strategy holds particular promise for treating metabolic disorders such as diabetes and lysosomal storage diseases, where enzymatic dysfunction leads to pathological accumulation of substrates.

A key advantage of DNA-based assembly is the ability to control inter-enzyme distances with nanometer precision. For example, glucose oxidase (GOx) and catalase (CAT) can be positioned on a DNA scaffold to create a two-step cascade for glucose metabolism. GOx converts glucose into gluconic acid and hydrogen peroxide, while CAT decomposes the cytotoxic hydrogen peroxide into water and oxygen. When these enzymes are free in solution, intermediate diffusion reduces efficiency and increases oxidative stress due to prolonged hydrogen peroxide exposure. In contrast, DNA-assembled nanofactories localize the reactions, minimizing harmful byproduct accumulation and enhancing overall kinetics. Studies have demonstrated that such systems achieve up to a fivefold increase in reaction rates compared to free enzyme mixtures.

The concept extends beyond diabetes therapy. Lysosomal storage diseases, caused by deficiencies in lysosomal enzymes, could benefit from DNA nanofactories that co-localize missing enzymes with auxiliary proteins. For instance, a Gaucher disease nanofactory might include beta-glucocerebrosidase alongside saposin C, a protein that enhances substrate binding. The DNA scaffold ensures optimal enzyme ratios and spatial arrangement, improving substrate processing and reducing aberrant accumulation of glucocerebroside.

Substrate channeling is a critical feature of these systems. By positioning enzymes at precise intervals, the product of one reaction becomes the substrate for the next with minimal diffusion losses. DNA origami structures, such as hexagonal or rectangular tiles, provide rigid frameworks to maintain these geometries. Computational modeling suggests that inter-enzyme distances below 10 nm maximize channeling efficiency, while larger separations revert to diffusion-limited kinetics. Experimental data supports this, showing that reaction yields drop significantly when enzymes are spaced beyond 20 nm apart.

Immunogenicity remains a concern for therapeutic applications. While DNA itself is less immunogenic than viral vectors or synthetic polymers, the attached enzymes or foreign DNA sequences could trigger immune responses. Strategies to mitigate this include using humanized enzymes or shielding the nanofactory with polyethylene glycol (PEG) coatings. Studies in murine models indicate that PEGylated DNA-enzyme constructs exhibit reduced antibody generation compared to unmodified versions, though long-term immune effects require further investigation.

Contrasting DNA-assembled nanofactories with free enzyme formulations highlights several advantages. Free enzymes suffer from rapid renal clearance, proteolytic degradation, and inefficient substrate processing due to random collisions. In vivo experiments show that DNA-scaffolded enzymes persist in circulation up to three times longer than free counterparts, attributed to the nanostructure’s larger hydrodynamic radius. Additionally, the localized activity of nanofactories reduces off-target effects. For example, free catalase administered with GOx may scavenge hydrogen peroxide systemically, interfering with redox signaling. DNA co-localization restricts this activity to the immediate vicinity of GOx, preserving physiological hydrogen peroxide gradients.

Challenges persist in scaling up production and ensuring stability under physiological conditions. DNA nanostructures can degrade in serum due to nuclease activity, though chemical modifications like phosphorothioate linkages improve resistance. Enzyme leakage from scaffolds is another issue; covalent attachment strategies have shown promise but may reduce enzymatic activity. Recent advances in click chemistry and affinity-based tagging offer solutions, with some systems retaining over 90% of enzymes after 72 hours in serum.

Future directions include integrating feedback mechanisms into nanofactories, such as glucose-sensitive DNA switches to modulate enzyme activity dynamically. Multi-enzyme cascades for complex metabolic pathways, like glycogen synthesis or cholesterol degradation, are also under exploration. The modularity of DNA assembly allows for rapid reconfiguration, enabling personalized therapies tailored to individual enzymatic deficiencies.

In summary, DNA-assembled nanofactories provide a versatile platform for metabolic therapy, combining the precision of nucleic acid nanotechnology with the catalytic power of enzymes. By addressing limitations of free enzyme formulations—such as poor stability, inefficient channeling, and immunogenicity—these systems open new avenues for treating metabolic diseases. Continued refinement of stability, scalability, and immune compatibility will be essential for clinical translation.