Team:Slovenia

nanoBRICKs[pro] – synthetic smart nanomaterials from nano to macro

Nanotechnology designs materials with advanced properties based on the control of molecular structure at the nanoscale. Biological systems provide an attractive opportunity to design and easily manufacture material with programmable properties. DNA origami demonstrated the power of this technology by creating a variety of assemblies that can be easily encoded in the nucleotide sequence. However, for biological nanodevices nature favors polypeptides over nucleic acids due to stability and versatility of amino-acid side chains. With few exceptions protein and peptide assemblies have been considered too difficult for the bottom-up design due to complex interactions and manufacturing problems specific for each case. We present technology for manufacturing nanomaterials based on combinations of modular peptide elements and protein domains, which allow self-assembly into complex tertiary structures with designed macroscopic properties. We demonstrate the feasibility and potentials of protein nanotechnology by design, streamlining the production and technological application of nanomaterials based on nanoBRICKsPRO.

Abstract for nonspecialists

Imagine that you could manufacture complex devices that self-assemble from their components, imagine that those components measure a few nanometers, imagine that you could have a factory that produces those devices from simple sugars or even from solar light and carbon dioxide. This is exactly what is going on in cells as the ultimate factories and the devices that are produced are mainly made of polypeptides, such as enzymes, silk or hair. We set to prepare and test modular genetic elements we named nanoBricks, to create self-assembling material to form structures unseen in nature. In comparison to usual nanomaterials we can program the composition of polypeptide nanomaterials through DNA code. This allows us an unrivaled control of their composition at nanoscale, which determines the properties of those materials. We achieved this by combining small building blocks, called coiled-coils that twist around each other, forming rigid rods. Several of those rods are linked by flexible hinges into chains, which can assemble into complex polyhedra and into planar and three-dimensional networks. Those assemblies may contain pores of a defined size, which we can modify at will with respect to size or chemical properties. We used nanoBricks to prepare functional ultrafiltration membrane and material that can be assembled or disassembled by the addition of small molecules. The high level of control at the nanoscale, ease and sustainability of production has exciting potentials for manufacturing sophisticated scaffolds, biomineralization, drug delivery and many, many more applications.

Figure 1: Modular nanoBricks were used to prepare polypeptide chain, composed of three designed coiled-coil -forming segments (shown as arrows), which guide the polypeptide chains into nanobox as well as into planar lattice (left), which we experimentally confirmed (right, transmission electron microscopic image).

Summary of our main achievements

• We designed the self-assembling polyhedra based on combinations of coiled-coil-forming segment as the polypeptide equivalent of DNA origami producing structures that do not exist in nature. We analyzed the topology of possible combinations of coiled-coil-forming segments and for the first time demonstrated the feasibility of this approach on a polypeptide composed of three designed coiled-coil-forming segments. This polypeptide can by slow annealing assemble a box or two-dimensional polypeptide lattice, depending on its concentration. Formation of nanoscale assembly and polypeptide lattice was confirmed by AFM and TEM.

• We prepared polypeptide assemblies, based on gyrase B fragment, which can be assembled and disassembled by the addition of different compounds, which has particularly useful properties for drug delivery.

• We proposed an approach for biomaterial design that could be crosslinked through coiled-coil interactions, which will allow introducing additional functional polypeptides that provide different functions to the cell-growth matrix, such as antimicrobial activity (LL-37 peptide), cell differentiation (NGF), growth factors etc, with an almost unlimited number of potential combinations.

• We prepared polypeptide network based on self-assembly of a polypeptide that contains a p53 tetramerization domain and a coiled coil domain. Real world application such as ultrafiltration was demonstrated on self-assembled polypeptide membrane for the removal of viruses.

• We introduced a BioBrick standard variant ([http://dspace.mit.edu/bitstream/handle/1721.1/46705/BBFRFC37.pdf?sequence=1 BBF RFC 37]) that allows modification of the length of the linker between polypeptide building blocks and prepared over 100 new BioBricks comprising different coiled-coil combinations, oligomerizing polypeptide domains and functional domains that are dedicated to the manufacturing of polypeptide nanoassemblies.

• We introduced a procedure that streamlines and unifies manufacturing of polypeptides which results, in principle independently of their sequence, in high yield polypeptide production in bacteria regardless of their toxicity, sensitivity to proteolysis and minimizes the purification steps.