(235c) Hybrid Protein-DNA and Peptide-DNA Nanostructures

Two of the most commonly used molecules for self-assembling nanotechnology are DNA and peptides/proteins. DNA nanotechnology has enabled the construction of complex structures with unparalleled addressability and anisotropy thanks to the predictable base-pairing rules and the well-understood physical properties of the double helix. This versatility, however, comes at the expense of chemical heterogeneity and limited functionality. By contrast, peptides and proteins have the advantage of expanded chemical diversity, diverse structural motifs, and biological relevance. However, most peptide/protein-based nanostructures are highly symmetric and not precisely addressable. The ability to merge the programmability of DNA nanotechnology with the chemical, structural, and functional diversity of proteins and peptides represents a powerful opportunity in bio-nanotechnology.

Herein, we describe our work merging self-assembling protein and peptide motifs with DNA nanostructures through the use of site-specific protein/peptide-DNA conjugates (Fig. 1). We report two systems: (1) tetrahedral cages comprised of a heterotrimeric protein linked to a triangular wireframe DNA base (Fig. 1A); and (2) a supramolecular polymer of DNA origami cuboids linked by coiled-coil peptides (Fig. 1B). In both cases, the protein-DNA or peptide-DNA conjugates are synthesized through chemical conjugation with unique reactive amino acids, and the resulting hybrids are used as addressable building blocks for subsequent, hierarchical assembly with DNA structures bearing complementary handles.

The tetrahedral protein-DNA cages are comprised of a highly stable homotrimeric aldolase protein modified with ssDNA handles.¹ We demonstrate the synthesis of cages comprised of both three and four helical turns of DNA (10 and 14 nm edge lengths, respectively), and demonstrate the three-dimensional structure using both AFM and indirect chemical characterization methods. We also demonstrate the use of two different site-specific coupling reactions, as well as the effect of modification site on cage assembly yield. The DNA components provide for unparalleled addressability of the cage, whereas the protein can provide novel structural, catalytic, or scaffolding properties (e.g. fusion with targeting protein/peptides).

For the coiled-coil/DNA system, we modify each pair of a heterodimeric coiled-coil pair with unique ssDNA handles.² We demonstrate coiled-coil formation and hierarchical assembly of DNA tiles and origami cuboid structures using the conjugates. We probe several different assembly protocols, including: one-pot formation of origami fibers, hierarchical formation of dimers/trimers/alternating copolymers, and polymerization of purified origami with a pre-formed coiled-coil/DNA building block in a second step. Importantly, the coiled-coil provides a novel self-assembly motif and molecular scaffold that is orthogonal to DNA hybridization, and will enable the incorporation of novel functionality. We also use bioactive fibronectin proteins modified with two orthogonal coils as “staples” that can assemble DNA structures without the need to chemically modify the protein with DNA.