DNA-Functionalized, Bivalent Proteins

Abstract

Bivalent DNA conjugates of β-galactosidase (βGal), having pairs of oligonucleotides positioned closely on opposing faces of the protein, have been synthesized and characterized. These structures, due to their directional bonding characteristics, allow for the programmable access of one-dimensional protein materials. When conjugates functionalized with complementary oligonucleotides are combined under conditions that support DNA hybridization, periodic wire-type superstructures consisting of aligned proteins form. These structures have been characterized by gel electrophoresis, cryo-transmission electron microscopy, and negative-stain transmission electron microscopy. Significantly, melting experiments of complementary building blocks display narrowed and elevated melting transitions compared to the free duplex DNA, further supporting the formation of the designed binding mode, and unambiguously characterizing their association as DNA-mediated. These novel structures illustrate, for the first time, that directional DNA bonding can be realized with only a pair of DNA modifications, which will allow one to engineer directional interactions and realize new classes of superstructures not possible simply through shape control or isotropically functionalized materials.

Programmable Atom Equivalents: Atomic Crystallization as a Framework for Synthesizing Nanoparticle Superlattices

“…First, while early work in PAE synthesis revealed multiple routes to either patchy or asymmetrically functionalized particles, the particles synthesized with these methods have not yet been demonstrated to form ordered crystals, and breaking the symmetry of spherical building blocks remains a challenge. Nevertheless, by functionalizing specific sites on pseudo‐spherical proteins with DNA, PAEs were synthesized with tunable and precise bond distributions where both the number and direction of DNA linkages were controlled . Such constructs have been demonstrated to produce arrangements that are unachievable with isotropically functionalized spheres .…”

“…Such constructs have been demonstrated to produce arrangements that are unachievable with isotropically functionalized spheres . It is even possible to functionalize different sites on the same protein with orthogonal DNA sticky ends, thereby yielding Janus‐type PAEs that assemble into 1D crystalline chains, or complex layered crystalline structures of PAEs that alternate in NP core identity (composition or size) (Section .). A second strategy to impart directional interactions between spherical particles involves trapping NPs within or at the vertices of anisotropic DNA origami constructs .…”

“…Finally, the PAEs with programmed valency and directional binding as a result of anisotropic core shape or surface functionalization (Section .) have exhibited several unique lattice arrangements not currently realized in PAEs with isotropic binding, namely 1D and 2D crystals, body‐centered tetragonal (bct) unit cells, layered structures, the Laves phase isostructural to dicopper magnesium (MgCu 2 ), and even diamond symmetry . As these more complex phases indicate, further experimental and theoretical investigations are required to garner a full understanding of the multiple different forces that are important in dictating PAE assembly.…”

Toward Precise Manipulation of DNA–Protein Hybrid Nanoarchitectures

“…Methods involving protein engineering offer more specificity and stoichiometric control over DNA attachment. Using a D 2 symmetric, tetrameric protein β‐galactosidase (β‐Gal) as a model protein, McMillan and Mirkin recently engineered β‐Gal with a cysteine mutation at residue T265 for conjugating maleimide‐terminated DNA on the top and bottom face of the protein tetramer . The DNA strand‐mediated orientation control facilitates alignment of β‐Gal axially into 1D DNA–protein nanowires instead of nonspecific aggregation ( Figure a).…”

“…a) Engineering of site‐specific conjugation of DNA to recombinant β‐Gal for controllable self‐assembly upon DNA–DNA interactions. Reproduced with permission . Copyright 2018, American Chemical Society.…”