Two-step nanocage: Individual enzymes (orange and green) are first attached to half-cage structures. Half cages are then assembled into full cages, where reactants are brought into close proximity. [Jason Drees: The Biodesign Institute at Arizona State University]

 

Cells and even smaller enclosures—nature’s tiny bioreactors—have long aroused envy among synthetic biologists. By compartmentalizing enzymes and substrates, these enclosures bring chemicals into close proximity while shielding them from degradation, allowing desirable reactions to proceed undisturbed by competing reactions at rates hardly limited by diffusion kinetics.

In the hope of bringing all these advantages to artificial compartments, synthetic biologists at Arizona State University have been assembling nanometer-scale cages. Actually, it would be more correct to say that they have been orchestrating the self-assembly of such cages, which consist of lengths of DNA.

“We have been designing programmable DNA nanostructures with increasing complexity for many years, and it is now time to ask what can we do with these structures,” said Hao Yan, Ph.D., director of the Center for Molecular Design and Biomimetics at Arizona State University's Biodesign Institute. “We have used designer DNA nanocages to compartmentalize enzymatic reactions in a confined environment. Drawing inspiration from nature, we have uncovered interesting properties, some unexpected.”

Dr. Yan led an effort to build nanocages that would have well-controlled architectures and enable the systematic study of how encapsulation conditions might affect a set of common metabolic enzymes. These conditions included “proximal polyanionic surfaces.” In addition to bringing enzyme-substrate pairs closer together, encapsulation could facilitate activity through the unique electrical charge density with the nanocage.

Dr. Yan’s team presented their work February 10 in the journal Nature Communications, in an article entitled “Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.” This article described how synthetic bioreactors were built to house enzymes and their substrates, allowing chemical conversions to take place in a controlled environment. Each minute structure, which measured just 54 nanometers across, was something like a Faberge egg whose separate halves fit together to encapsulate their chemical contents.

“Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes,” wrote the authors. “Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer.”

The construction of the nanocages took place in two steps. First, individual enzymes were attached to open half-cage structures. Then, the half-cages were fitted together into a full, closed nanocage. To create the half-cages, a technique known as DNA origami was used. Lengths of viral DNA are prepared to self-assemble into a honeycomb lattice, via the pairing of A and C nucleotides and of C and T nucleotides.

The open-sided half-cages of the DNA nanocages allow the access of large protein molecules into the nanocage's internal cavity. The two half-cages are fitted together with the aid of short bridge DNA strands that bind with complementary DNA sequences extending from the edges of either half-cage, (see accompanying animation). The small gaps on each of the top and bottom surfaces of the DNA nanocage allow the diffusion of small molecules across the DNA walls.

To examine the resulting structures, transmission electron microscopy was used, along with gel electrophoresis and single molecule fluorescence experiments, which demonstrated that close to 100% of the DNA segments properly formed half-cage structures and more than 90% formed full cages.

The study examined six different enzymes, ranging in size from the smallest, which measured ~44kD (kilodaltons) to the largest, ~ 450 kD. All six enzymes were successfully encapsulated in nanocages, though the yields varied according to enzyme size. The largest enzyme examined, known as β-galactosidase, showed the lowest yield of 64%.

An evaluation of enzyme activity showed a four- to tenfold increase for enzymes encapsulated in nanocages, compared with the activity of free enzymes. Enzyme turnover rate—defined as the maximum number of chemical conversions of substrate molecules per second—was inversely correlated with the size of encapsulated enzymes, with the smallest enzyme yielding the highest turnover.

“We further show that DNA nanocages protect encapsulated enzymes against proteases,” note the authors of the Nature Communications article, “demonstrating their practical utility in functional biomaterials and biotechnology.”