DNA-mediated engineering of multicomponent enzyme crystals

Abstract

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein-protein interactions for DNA-DNA interactions to direct the assembly of two nucleic-acid-functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA-DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.

Keywords: DNA-programmable assembly; biomaterials; nanoscience; self-assembly; superlattice.

Fig. 1.

Synthesis and characterization of protein–DNA conjugates. (A) Cartoon depictions of bovine and Cg catalases showing their molecular topologies and the locations of surface-accessible amines (blue sticks). (B) Scheme for the synthesis and assembly of DNA-functionalized catalases. Surface-accessible amines were modified with azides containing NHS and N₃ moieties at opposing termini (i), after which the covalently attached azides were conjugated to two distinct 5′-DBCO–modified DNA strands via a copper-free “click chemistry” reaction (ii). Hybridization of linker strands to the DNA-functionalized proteins (iii) followed by mixing of proteins with complementary linkers (iv) results in the assembly of the proteins into BCC or CsCl-type unit cells. (C–E) Comparison of the hydrodynamic diameters of native (C), N₃-functionalized (D), and DNA-functionalized (E) Cg catalases, as determined by DLS. (F) Comparison of the enzyme-catalyzed rates of the disproportionation of H₂O₂ as a function of substrate concentration by native (black circles), DNA-functionalized [red (strand 1) and blue (strand 2) squares], and crystalline (cyan triangles) Cg catalase. (G) Thermal melting transition of DNA-templated aggregates composed of Cg catalase.

Fig. 2.

SAXS data for protein–protein (A–D) and protein–AuNP (E and F) superlattices. Each panel shows a comparison between the experimentally observed 1D SAXS patterns (red) and theoretical predictions (black or cyan traces) for protein-containing superlattices. A schematic representation of the components and unit cell of each superlattice type is shown at the Top of each panel, where Cg catalase, bovine catalase, and AuNPs are depicted as a red cartoon, cyan cartoon, or gold sphere, respectively. The superlattices are isostructural with (A) a mixture of BCC (blue theoretical trace) and CsCl (black theoretical trace), (B–D) CsCl, and (E and F) simple cubic.

Fig. 3.

Characterization of single crystalline superlattices by TEM. Low (A)- and high (B)-magnification TEM micrographs of Cg catalase–AuNP hybrid superlattices showing the uniform formation of the expected rhombic dodecahedron crystal habit. Cartoon depictions of the various orientations of a rhombic dodecahedron are shown side-by-side with experimentally observed superlattices with matching orientations. The Inset in B depicts the high degree of short-range order between AuNPs within a single crystal. (C and D) Low- and high-magnification TEM images of superlattices composed of Cg catalase. (D) A high-magnification TEM image of a single Cg catalase crystal with clearly visible lattice planes demonstrating its single crystalline nature. (Scale bars: 5 µm for A and C, 500 nm for B, and 200 nm for D).