Effect of Surfactant Concentration and Hydrophobicity on the Ordering of Water at a Silica Surface.

The performance of nonionic surfactants is mediated by the interfacial interactions at the solid-liquid interface. Here we applied sum frequency generation (SFG) vibrational spectroscopy to probe the molecular structure of the silica-nonionic surfactant solution interface in situ, supplemented by quartz crystal microbalance with dissipation monitoring (QCM-D) and molecular dynamics (MD) simulations. The combined studies elucidated the effects of nonionic surfactant solution concentration, surfactant composition, and rinsing on the silica-surfactant solution interfacial structure. The nonionic surfactants studied include ethylene-oxide (EO) and butylene oxide (BO) components with different ratios. It was found that the CH groups of the surfactants at the silica-surfactant solution interfaces are disordered, but the interfacial water molecules are ordered, generating strong SFG OH signals. Solutions with higher concentrations of surfactant lead to a slightly higher amount of adsorbed surfactant at the silica interface, resulting in more water molecules being ordered at the interface, or a higher ordering of water molecules at the interface, or both. MD simulation results indicated that the nonionic surface molecules preferentially adsorb onto silanol sites on silica. A surfactant with a higher EO/BO ratio leads to more water molecules being ordered and a higher degree of ordering of water molecules at the silica-surfactant solution interface, exhibiting stronger SFG OH signal, although less material is adsorbed according to the QCM-D data. A thin layer of surfactants remained on the silica surface after multiple water rinses. To the best of our knowledge, this is the first time the combined approaches of SFG, QCM-D and MD simulation techniques have been applied to study nonionic surfactants at the silica-solution interface, which enhances our understanding on the interfacial interactions between nonionic surfactants, water and silica. The knowledge obtained from this study can be helpful to design the optimal surfactant concentration and composition for future applications.

DNA-Directed Protein Packing within Single Crystals.

Designed DNA-DNA interactions are investigated for their ability to modulate protein packing within single crystals of mutant green fluorescent proteins (mGFPs) functionalized with a single DNA strand (mGFP-DNA). We probe the effects of DNA sequence, length, and protein-attachment position on the formation and protein packing of mGFP-DNA crystals. Notably, when complementary mGFP-DNA conjugates are introduced to one another, crystals form with nearly identical packing parameters, regardless of sequence if the number of bases is equivalent. DNA complementarity is essential, because experiments with non-complementary sequences produce crystals with different protein arrangements. Importantly, the DNA length and its position of attachment on the protein markedly influence the formation of and protein packing within single crystals. This work shows how designed DNA interactions can be used to influence the growth and packing in X-ray diffraction quality protein single crystals and is thus an important step forward in protein crystal engineering.

Protein Materials Engineering with DNA.

Proteins are a class of nanoscale building block with remarkable chemical complexity and sophistication: their diverse functions, shapes, and symmetry as well as atomically monodisperse structures far surpass the range of conventional nanoparticles that can be accessed synthetically. The chemical topologies of proteins that drive their assembly into materials are central to their functions in nature. However, despite the importance of protein materials in biology, efforts to harness these building blocks synthetically to engineer new materials have been impeded by the chemical complexity of protein surfaces, making it difficult to reliably design protein building blocks that can be robustly transformed into targeted materials. Here we describe our work aimed at exploiting a simple but important concept: if one could exchange complex protein-protein interactions with well-defined and programmable DNA-DNA interactions, one could control the assembly of proteins into structurally well-defined oligomeric and polymeric materials and three-dimensional crystals. As a class of nanoscale building block, proteins with surface DNA modifications have a vast design space that exceeds what is practically and conceptually possible with their inorganic counterparts: the sequences of the DNA and protein and the chemical nature and position of DNA attachment all play roles in dictating the assembly behavior of protein-DNA conjugates. We summarize how each of these design parameters can influence structural outcome, beginning with proteins with a single surface DNA modification, where energy barriers between protein monomers can be tuned through the sequence and secondary structure of the oligonucleotide. We then explore challenges and progress in designing directional interactions and valency on protein surfaces. The directional binding properties of protein-DNA conjugates are ultimately imposed by the amino acid sequence of the protein, which defines the spatial distribution of DNA modification sites on the protein. Through careful design and mutagenesis, bivalent building blocks that bind directionally to form one-dimensional assemblies can be realized. Finally, we discuss the assembly of proteins densely modified with DNA into crystalline superlattices. At first glance, these protein building blocks display crystallization behavior remarkably similar to that of their DNA-functionalized inorganic nanoparticle counterparts, which allows design principles elucidated for DNA-guided nanoparticle crystallization to be used as predictive tools in determining structural outcomes in protein systems. Proteins additionally offer design handles that nanoparticles do not: unlike nanoparticles, the number and spatial distribution of DNA can be controlled through the protein sequence and DNA modification chemistry. Changing the spatial distributions of DNA can drive otherwise identical proteins down distinct crystallization pathways and yield building blocks with exotic distributions of DNA that crystallize into structures that are not yet attainable using isotropically functionalized particles. We highlight challenges in accessing other classes of architectures and establishing general design rules for DNA-mediated protein assembly. Harnessing surface DNA modifications to build protein materials creates many opportunities to realize new architectures and answer fundamental questions about DNA-modified nanostructures in both materials and biological contexts. Proteins with surface DNA modifications are a powerful class of nanomaterial building blocks for which the DNA and protein sequences and the nature of their conjugation dictate the material structure.

Programming Protein Polymerization with DNA.

A strategy that utilizes DNA for controlling the association pathway of proteins is described. This strategy uses sequence-specific DNA interactions to program energy barriers for polymerization, allowing for either step-growth or chain-growth pathways to be accessed. Two sets of mutant green fluorescent protein (mGFP)-DNA monomers with single DNA modifications have been synthesized and characterized. Depending on the deliberately controlled sequence and conformation of the appended DNA, these monomers can be polymerized through either a step-growth or chain-growth pathway. Cryo-electron microscopy with Volta phase plate technology enables the visualization of the distribution of the oligomer and polymer products, and even the small mGFP-DNA monomers. Whereas cyclic and linear polymer distributions were observed for the step-growth DNA design, in the case of the chain-growth system linear chains exclusively were observed, and a dependence of the chain length on the concentration of the initiator strand was noted. Importantly, the chain-growth system possesses a living character whereby chains can be extended with the addition of fresh monomer. This work represents an important and early example of mechanistic control over protein assembly, thereby establishing a robust methodology for synthesizing oligomeric and polymeric protein-based materials with exceptional control over architecture.

DNA-Encoded Protein Janus Nanoparticles.

Asymmetric functionality and directional interactions, which are characteristic of noncentrosymmetric particles, such as Janus particles, present an opportunity to encode particles with properties, but also a great synthetic challenge. Here, we exploit the chemical anisotropy of proteins, and the versatile chemistry of DNA to synthesize a protein-based Janus nanoparticle comprised of two proteins encoded with sequence-specific nucleic acid domains, tethered together by an interprotein "DNA bond". We use these novel nanoparticles to realize a new class of three-dimensional superlattice, only possible when two sides of the particle are modified with orthogonal oligonucleotide sequences. The low symmetry, intrinsic to Janus particles, enables the realization of unprecedented multicomponent nanoparticle superlattices with unique, hexagonal layered architectures. In addition, the interprotein "DNA bond" can be modulated to selectively expand the lattice in a single direction. The results presented herein not only emphasize the power of rationally designing nanoscale building blocks to create highly engineered colloidal crystals, but also establish a precedent for applications of multidomain DNA-encoded nanoparticles, especially in the field of colloidal crystallization.

DNA-Functionalized, Bivalent Proteins.

Bivalent DNA conjugates of ß-galactosidase (ßGal), 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.

Modulating Nanoparticle Superlattice Structure Using Proteins with Tunable Bond Distributions.

Herein, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using beta-galactosidase (ßgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When these are assembled into crystalline superlattices with gold nanoparticles, we find that the distribution of DNA modifications modulates the favored structure: ßgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB2 packing. We probe the role of protein oligonucleotide number and conjugate size on this observation, which revealed the importance of oligonucleotide distribution in this observed assembly behavior. These results indicate that proteins with defined DNA modification patterns are powerful tools for controlling nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.

DNA-Mediated Cellular Delivery of Functional Enzymes.

We report a strategy for creating a new class of protein transfection materials composed of a functional protein core chemically modified with a dense shell of oligonucleotides. These materials retain the native structure and catalytic ability of the hydrolytic enzyme ß-galactosidase, which serves as the protein core, despite the functionalization of its surface with ∼25 DNA strands. The covalent attachment of a shell of oligonucleotides to the surface of ß-galactosidase enhances its cellular uptake of by up to ∼280-fold and allows for the use of working concentrations as low as 100 pM enzyme. DNA-functionalized ß-galactosidase retains its ability to catalyze the hydrolysis of ß-glycosidic linkages once endocytosed, whereas equal concentrations of protein show little to no intracellular catalytic activity.