Put functional proteins in their place



UPTON, NY – Scientists have organized proteins – nature’s most versatile building blocks – into desired 2D and 3D ordered arrangements while maintaining their structural stability and biological activity. They built these designer functional protein arrays using DNA as a programmable construction material. The team, representing the US Department of Energy’s Brookhaven National Laboratory (DOE), Columbia University, the DOE’s Lawrence Berkeley National Laboratory, and the City University of New York (CUNY) described its approach in the Jan. Nature communication.

“For decades, scientists have dreamed of assembling proteins rationally into specific organizations with preserved protein function,” said corresponding author Oleg Gang, head of the Soft and Bio Nanomaterials Group at the Center for Functional Nanomaterials (CFN) at the Brookhaven Lab and professor of chemical engineering and science for applied physics and materials science at Columbia Engineering. “Our DNA-based platform has enormous potential not only for structural biology, but also for various biotechnological, biomedical and bionanomaterial applications.”

The main motivation of this work was to find a rational way to organize proteins into designed 2-D and 3-D architectures while maintaining their function. The importance of the organization of proteins is well known in the protein crystallography art. In this technique, proteins are removed from their native solution-based environment and condensed into an ordered arrangement of atoms (crystalline structure), which can then be structurally characterized. However, because of their flexibility and aggregation properties, many proteins are difficult to crystallize, which requires trial and error. The structure and function of proteins can change during the crystallization process, and they can become inoperable during crystallization using conventional methods. This new approach opens up many possibilities for the production of engineered biomaterials beyond the goals of structural biology.

“The ability to produce biologically active protein lattices is relevant for many applications including tissue engineering, multi-enzyme systems for biochemical reactions, large-scale protein profiling for precision medicine, and synthetic biology,” added first author Shih-Ting (Christine) Wang, postdoc in the CFN Soft and Bio Nanomaterials Group.

Although DNA is best known for its role in storing our genetic information, the same base-pairing processes that are used for that storage can be used to construct the desired nanostructures. A single strand of DNA is made up of subunits, or nucleotides, of which there are four types (known by the letters A, C, T, and G). Each nucleotide has a complementary nucleotide that it attracts and binds to (A with T and C with G) when two strands of DNA are close together. Using this concept in the technique of DNA origami, scientists mix several short strands of synthetic DNA with a single long strand of DNA. The short strands bind to the long strand and “fold” it into a specific shape based on the sequence of bases that scientists can determine.

In this case, the scientists created octahedral-shaped DNA origami. Within these cage-like scaffolds, they placed strands of DNA with a specific “color” or coding sequence at specific locations (center and outside the center). They attach complementary strands of DNA to the surface of proteins – especially ferritin, which stores and releases iron, and apoferritin, its iron-free counterpart. By mixing the DNA cages and conjugated proteins and heating the mixture to promote the reaction, the proteins were delivered to the internal designated sites. They also created empty cages with no protein inside.

In order to connect these nanoscale building blocks or protein voxels (DNA cages with encapsulated proteins) in the desired 2-D and 3-D arrays, the co-author and Columbia doctoral student Brian Minevich designed different colors for the external bonds of the voxels . With this color scheme, the voxels would recognize each other in a programmable, controllable manner, which would lead to the formation of specially prescribed types of protein grids. To demonstrate the versatility of the platform, the team constructed single- and double-layer 2D arrays as well as 3D arrays.

“By arranging the colors in a certain way, we can program the formation of different grids,” explains Gang. “We have full control over the design and construction of the desired protein lattice architectures.”

To confirm that the proteins were encapsulated in the cages and that the grids were constructed as planned, the team turned to various electron- and X-ray-based imaging and scattering techniques. These techniques included electron microscopy (EM) imaging at the CFN; Small-angle X-ray scattering at the National Synchrotron Light Source II (NSLS-II) Complex Materials Scattering (CMS) and Life Science X-ray Scattering (LiX) beamlines in Brookhaven; and cryogenic EM imaging at Lawrence Berkeley’s Molecular Foundry (MF) and the CUNY Advanced Science Research Center. The CFN, NSLS-II and MF are all user facilities of the DOE Office of Science; CFN and MF are two of five DOE Nanoscale Science Research Centers.

“The science was made possible by advanced synthesis and characterization capabilities at three user facilities within the national laboratory system and one university facility,” said Gang. “Without these facilities and the expertise of the scientists from each of them, this study would not have been possible.”

Following these postgraduate studies, they investigated the biological activity of ferritin. By adding a reducing agent to the ferritin grid, they induced the release of iron ions from the center of the ferritin proteins.

“By monitoring the evolution of the SAXS patterns during iron release, we were able to quantify how much iron was released and how quickly it was released, as well as confirm that the integrity of the lattice was maintained during this protein operation,” Minevich said. “According to our TEM studies, the proteins stayed within the framework.”

“We have shown that the proteins can perform the same function as in a biological environment, while maintaining the spatial organization we created,” explains Wang.

Next, the team will apply its DNA-based platform to other types of proteins, with the goal of building more complex, functional protein systems.

“This research represents an important step in bringing together different components from real biological machines and organizing them into desired 2-D and 3-D architectures to create engineered and bioactive materials,” said Gang. “It’s exciting because we see the rational way to produce desired functional bio-nano-systems that nature has never produced before.”


This work was supported by the DOE Office of Science, a grant from the Laboratory Directed Research and Development Program, and the National Science Foundation. The LiX beamline is part of the Life Science Biomedical Technology Research resource, which is co-funded by the National Institute of General Medical Sciences and the DOE Office of Biological and Environmental Research with additional support from the National Institutes of Health. Brookhaven’s Biology Department supplied the proteins.

Brookhaven National Laboratory is supported by the US Department of Energy’s Office of Science. The Office of Science is the largest single funder of basic research in the physical sciences in the United States, working to address some of the most pressing challenges of our time. Further information can be found at https: ///Energy.Government/science.

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