Protein Materials Engineering with DNA | Accounts of Chemical

Article Cite This: Acc. Chem. Res. 2019, 52, 1939−1948

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Protein Materials Engineering with DNA Janet R. McMillan,†,∥ Oliver G. Hayes,†,∥ Peter H. Winegar,†,∥ and Chad A. Mirkin*,†,∥ Department of Chemistry and ∥International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States

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CONSPECTUS: 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.

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INTRODUCTION

polypeptides composed of a subset of 20 standard amino acids folded into defined and monodisperse 3D structuresare the central nanoscale building blocks of living systems. As individual building blocks, proteins can be remarkably homogeneous, yet as a class of building blocks, they are incredibly diverse in terms of their composition, structure, and

Controlling the bottom-up assembly of nanoscale building blocks into well-defined architectures is an important and unmet challenge in chemistry and materials science. The most impressive and complex examples of this are found in nature, where the assembly of proteins into higher-order structures, such as multiprotein oligomers,1,2 one-dimensional (1D) actin filaments,3,4 and three-dimensional (3D) collagen networks,5 is central to the function of biological systems. Proteins © 2019 American Chemical Society

Received: April 3, 2019 Published: June 14, 2019 1939

DOI: 10.1021/acs.accounts.9b00165 Acc. Chem. Res. 2019, 52, 1939−1948

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Accounts of Chemical Research function. Over millions of years, proteins have evolved welldefined surfaces that drive their assembly into higher-order materials,6,7 ranging from oligomeric complexes such as photosystems and cellulosomes2 to dynamic polymers such as actin and tubulin3,4,8 and other functional 2D and 3D architectures such as bacterial S-layers and collagen.5,9 Unfortunately, the chemical complexity of proteins and their surfaces makes it difficult to use this incredible class of nanoscale building blocks synthetically to build non-natural materials with structures and properties that are useful.10 Over the past two decades, remarkable progress has been made in addressing this challenge through the computational design of protein interfaces11−19 and in introducing controlled molecular interactions on protein surfaces, ranging from early work using biotin−streptavidin15 and protein−ligand20 interactions, to more recent advances that have demonstrated the utility of hydrophobic21,22 and host−guest23 interactions, and metal coordination chemistry24−29 in the design of protein materials. Despite this progress, control over protein assembly remains far behind what is possible with the sequenceprogrammable biopolymer, DNA.30,31 Unlike polypeptides, DNA has a more manageable polymer code based upon predictable, designable, and highly reliable Watson−Crick base-pairing interactions. Consequently, DNA has become a powerful tool for programming the crystallization of nucleic acid-modified nanoparticle building blocks32−39 and in designing precisely folded discrete and extended nanostructures.31,40−42 Since the first report of using DNA to program the assembly of gold nanoparticles (AuNPs) into macroscopic materials,32 the field has been refined to now include textbook methods for engineering colloidal crystals from a wide variety of DNAmodified constructs.39 The DNA-mediated crystallization of nanoparticles can be predicted and controlled using the complementary contact model (CCM), which operates under the premise that the crystal structure that maximizes the total number of DNA hybridization events will form. On the basis of this model, the DNA sequence, hydrodynamic particle size, DNA number, and particle shape can all be tuned to control the equilibrium crystal structure of a system (Figure 1).36 Proteins, however, offer design handles that nanoparticles do not: their chemically diverse, mutable, and well-defined surface chemistries allow the spatial distribution, number, and type of DNA strands to be tuned with precision not currently possible with inorganic particles (Figure 1). Expanding the principles developed to control the crystallization of nanoparticles to proteins provides both a creative solution to circumvent the challenges of designing protein interactions and exciting opportunities to explore a new colloidal crystal design space with the unique structural and chemical properties of proteins. From early work reporting the synthesis of covalent protein−DNA conjugates,43 several approaches have been taken to impart proteins with the programmable association properties of DNA in order to control their assembly into higher-order architectures. These efforts can be categorized into two classes: template-directed approaches, where DNA nanostructures define positions onto which proteins are immobilized, and surface-directed approaches, where the collective structural properties of protein surfaces modified with DNA dictate the assembly outcome. Early work on using DNA to organize proteins was limited to the use of singlestranded DNA templates,31,44−47 followed by the synthesis of more sophisticated discrete and extended structures based on

Figure 1. Comparison of the design parameters between inorganic nanoparticles and proteins.

DNA tiles,48−51 cages,52 or origami scaffolds.53−56 Other ordered, extended materials have been synthesized using interactions between nucleic acids and proteins with DNAbinding domains57 or through the combination of DNA hybridization and protein−protein interactions.58 Compared with template-based approaches, until recently there has been comparatively little progress in developing surface-based strategies.59−61 In spite of this limited activity, the impressive control that has been demonstrated using colloidal crystal engineering strategies with DNA establishes a blueprint for accessing protein materials unattainable with simple templatebased strategies.31,39 In this Account, we summarize recent work by our group and others aimed at developing methods to control the structure of protein-based materials using oligonucleotide surface modifications and directionality imposed by the shape of the protein or steric and electrostatic crowding of the DNA (Figure 2). Proteins with surface DNA modifications collectively form a new class of synthetically programmable building blocks where the amino acid sequence of the protein in concert with the sequence and chemical conjugation strategy of the oligonucleotides can be used to guide protein assembly and the ultimate structural outcome of the system.

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PROTEIN POLYMER ENGINEERING WITH DNA

DNA Sequence Controls Energy Barriers between Proteins

Proteins functionalized with a single DNA strand represent the simplest building blocks that we can consider. While a single DNA modification does not enable surface-directed assembly, the ability to program the sequence of the oligonucleotide permits a multitude of different interaction possibilities to be encoded.48,62 Inspired by the complex regulation processes of protein polymerization that govern cellular structure and 1940


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Accounts of Chemical Research

Figure 2. Proteins with surface DNA modifications that assemble into polymers and crystals. (A, B) Green fluorescent protein (GFP) with a single DNA modification in either a (A) single-stranded or (B) hairpin conformation. (C) β-Galactosidase (βGal) with four DNA modifications. (D) GroEl with 14 DNA modifications on each opposing face. (E) Catalase and (F) βGal with surface lysines functionalized with DNA. (G) DNAfunctionalized virus particle. (H) βGal with surface cysteines modified with DNA. (I) GFP-based Janus particle. Blue at the protein−DNA interface indicates that lysines are modified; red indicates that cysteines are modified. Inset cartoons show simplified building blocks and are not to scale.

function in nature,3,4,8 we set out to determine whether the sequence and structure of DNA could be used to influence the kinetic pathways of DNA-mediated protein polymerization. To conjugate single DNA strands to the surface of proteins, cysteine residues are a convenient choice because they can be easily introduced through genetic mutations, are not naturally present in high abundance, and can be functionalized using a variety of bioconjugation strategies.63 As a model system to study how DNA structure can be used to influence protein polymerization, we focused on monomeric green fluorescent protein (GFP), which can be easily tracked by its chromophore. Specifically, we prepared two sets of DNAmonofunctionalized GFP in which one DNA was singlestranded and the other was identical in length but had small sequence changes that allowed it to fold into a hairpin conformation.64 With these two sets of monomers, the effect of the DNA secondary structure on the polymerization of the protein−DNA monomers was studied. In both cases, polymerization is driven by a staggered complementary overlap between two halves of each of the 48 base pair (bp) DNA sequences between each pair of monomers (Figure 3A−D). Analytical size exclusion profiles along with gel electrophoresis characterization provided strong evidence that these GFP− DNA monomers were molecularly well-defined. For hairpin GFP−DNA monomers, 2D class averages of cryogenic transmission electron microscopy (cryo-TEM) micrographs proved to be a powerful characterization tool that showed welldefined electron density for both the protein and the appended DNA hairpin (Figure 3D).65 The combination of complementary single-stranded monomers resulted in their spontaneous polymerization into linear and cyclic structures, visualized by cryo-TEM, in a process similar to step-growth polymerization (Figure 3C,E). The combination of complementary GFP−hairpin DNA monomers resulted in a metastable mixture where proteins polymerized only upon the addition of an initiator strand that could open one set of hairpins and induce a cascade of protein polymerization, akin to chain-growth polymerization reactions (Figure 3D).64 The degree of polymerization of these products could be controlled by the concentration of initiator strand added, where a decrease in initiator stoichiometry led to products with higher apparent molecular weight, and as in

Figure 3. Control of the assembly pathway of proteins between stepand chain-growth by alteration of the conformation of surface DNA modifications. (A, B) DNA designs for (A) a single-stranded DNA system and (B) a hairpin DNA system. (C) Step-growth polymerization monomers and products. (D) Chain-growth monomers and products. The inset micrograph shows a 2D particle reconstruction of a chain-growth monomer. (E−G) Cryo-TEM micrographs of (E) step-growth products, (F) chain-growth products with low [initiator], and (G) chain-growth products with high [initiator]. Scale bars = 50 nm. Adapted from ref 65. Copyright 2018 American Chemical Society.

molecular-scale living polymerizations, polymers could be chain-extended with the addition of fresh monomer (Figure 3F,G).65 Therefore, this advance enables control over protein polymer molecular weight and structure between small linear and cyclic oligomers and significantly longer polymers, depending on the monomer concentration in the case of the 1941


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Accounts of Chemical Research step-growth system and the initiator stoichiometry in the case of the chain-growth system. The sequence and the conformation of DNA encoded therein represent a design handle to program energetic barriers to protein association in a manner reminiscent of strategies in supramolecular chemistry that manipulate the polymerization pathway of small molecules by designing kinetic barriers.66,67 Programming energy barriers between protein association events is something nature does with incredible elegance. In contrast, for other chemical strategies used to direct protein assembly, manipulating these barriers is more complicated than simply changing the sequence of a DNA strand. With this programmability, DNA-based strategies that allow chain growth of protein monomers may be employed to establish a new biomaterials space in which the physical and chemical properties of a material can be modulated by controlling the polymerization outcome.65 Protein Sequence and Structure Directs Valency

Energetic barrier control based on DNA sequence and structure provides a unique way of directing and controlling protein assembly. Designing higher-order protein materials or linear protein assemblies with well-defined directional binding between proteins, however, requires multiple DNA modifications to be introduced on the surfaces of proteins. Directional binding between DNA-functionalized nanostructures has been realized in the context of anisotropic nanoparticle crystallization35,68 as well as the assembly of small-molecule and polymer-DNA conjugates,69−71 where the multivalent nature of DNA binding imposes a thermodynamic driving force for directional assembly. In moving beyond proteins with single DNA modifications, we can draw inspiration from that work to design protein−DNA conjugates with a preference to interact in a directional manner. To realize such protein−DNA conjugates, DNA strands can be placed at desired points on a protein’s surface by controlling their chemical topology through genetic mutation. In other words, the directional binding properties and valency of protein−DNA conjugates are ultimately controlled by the underlying amino acid sequence and shape of the protein: these determine the spatial distribution of reactive residues for DNA conjugation, which in turn dictate the binding properties and thus the assembly outcome of a given conjugate (Figure 4A). Pioneering work by the Aida group realized directional interactions on opposing ends of the C7-symmetric chaperone protein GroEl through the multivalent interaction of 14 short (10 bp) sequences on each face. These sequences were conjugated through two surface cysteine mutations that were positioned closely on each subunit, which resulted in a protein that by symmetry had a ring of 14 DNA modifications on opposing faces. Negative-stain TEM of the products resulting from the combination of proteins functionalized with complementary DNA, after thermal annealing, revealed that these building blocks assembled in a highly directional manner into linear structures (Figure 4B,C). Interestingly, these interactions could be reversed through the addition of a specific oligonucleotide designed to displace the base pairing between proteins.72 Proteins that do not possess high-order cyclic symmetry make it difficult to place a comparable number of DNA modifications on their faces. However, the cooperative DNA hybridization that drives the directional assembly of GroEl is possible with far fewer numbers of DNA strands. DNA-

Figure 4. Valency of proteins can be dictated through the surface chemical topology and designed to realize directional interactions of protein surfaces. (A) Protein amino acid sequence ultimately dictates protein valency. (B) βGal (top) and GroEl (bottom) functionalized on opposing faces assemble into 1D materials. (C) Negative-stain TEM image of linear GroEl assemblies. Scale bar = 200 nm. Adapted from ref 72. Copyright 2017 American Chemical Society. (D) Negative-stain TEM image of linear βGal assemblies. Scale bar = 100 nm. Adapted from ref 73. Copyright 2018 American Chemical Society.

mediated directional protein assembly has been realized using only a pair of DNA modifications closely positioned on D2symmetric β-galactosidase (βGal). These conjugates with four DNA strands on their surfaces display DNA-mediated melting transitions that are significantly elevated relative to that of the free DNA under identical experimental conditions, which is suggestive of cooperative hybridization between the pairs of DNA strands on each face of the protein. These building blocks assemble into 1D structures through DNA hybridization (Figure 4B,D).73 These examples of programming directional interactions and valency into proteins with DNA rely on the amino acid sequence and structure of the protein to define the spatial distribution of DNA on its surface and thus the binding properties of the conjugate. The work that has been done so far points to the potential for a much larger design space that can be explored to access other types of protein materials and the need for more fundamental studies that elucidate the geometric, flexibility, and DNA length requirements that are essential for driving directional protein assembly. 1942


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protein interactions.33 Interestingly, icosahedral virus−DNA conjugates studied with complementary AuNPs in the latter example formed structures with very short range ordering that the authors claimed correspond to NaTl interpenetrating diamond lattices rather than the expected CsCl arrangement, which was explained to result from core particle interactions.59 To more thoroughly investigate how densely modified proteins behave in the context of DNA-mediated crystallization, catalase, a tetrameric D2-symmetric protein, was selected as a model system because of its stability and the relatively even distribution of lysine residues on its surface. Upon DNA functionalization and hybridization of complementary linker strands, catalase conjugates displayed highly cooperative and elevated melting transitions, indicative of the multivalent nature of DNA binding on the protein surface. When assembled either with a protein (of the same or a different variant of catalase) or AuNPs functionalized with complementary DNA, CsCl arrangements were observed in all cases (Figure 6A−C), where the resulting crystals retain the

PROTEIN CRYSTAL ENGINEERING WITH DNA

Crystallization of Isotropically Functionalized Proteins

So far, we have discussed the assembly of proteins with discrete numbers of DNA modifications. We have learned that the sequence of DNA can dictate the pathway of polymerization in proteins with only a single modification and that the sequence of a protein can be manipulated to design building blocks that can interact with programmed directionality and valency. To move beyond bivalent proteins and realize 3D architectures, larger numbers of oligonucleotides are required to impart sufficient multivalency to drive interactions in three dimensions. While cysteine residues are ideal handles for site-specific conjugation of small numbers of DNA strands, to modify proteins with more than a handful of oligonucleotides, surface lysine residues offer a convenient chemical handle, since most proteins naturally have a high density of these residues on their surface. Like thiols, amines can be easily modified using high-yielding reactions whose conditions are compatible with protein stability, such as a combination of Nhydroxysuccinimide ester and click chemistry or a host of other bioconjugation reactions.59,60,74,75 Nanoscale particles with surfaces densely functionalized with nucleic acids assemble as predicted by the CCM, according to which the structure that maximizes the number of DNA base-pairing interactions will form when a system is given sufficient thermal energy to reorganize to its thermodynamic product.36 This principle dictates that in a system of two complementary particles, a lattice isostructural with CsCl will form, as this is the structure that allows for the most contact area between complementary particles.36 These rules, therefore, can serve as guiding design principles for protein crystal engineering with DNA. The first examples of surface-driven DNA-mediated protein assembly focused on the assembly of isotropically functionalized viruses, first into disordered protein-only 3D structures60 and subsequently into moderately ordered lattices with complementary AuNPs.59 A key difference in moving from disordered protein aggregates to crystalline superlattices lies in the use of DNA linker strands that are fully complementary to the surface strand except for short (4−10 bp) “sticky ends” that enable lattice reorganization (Figure 5B), as opposed to the direct hybridization of the 20 bp surface strand (Figure 5A), where reorganization is prohibited at experimentally relevant temperatures because of the multivalent nature of

Figure 6. (A) Catalase densely functionalized with DNA assembles into CsCl crystals with complementary (B) catalase or (C) nanoparticles. (B, C) TEM images of catalase-only and binary catalase−AuNP crystals. Scale bars = (left) 200 nm, (right) 250 nm, (inset) 100 nm. Adapted with permission from ref 74. Published by the National Academy of Sciences. (D) Representation of a poly(ethylene glycol) (PEG) spacer between DNA and the catalase surface where n is the number of PEG spacers. (E) TEM image of CsCl catalase−AuNP crystals. (F) TEM image of thorium phosphide catalase−AuNP crystals when all of the PEG spacers are removed. In (E) and (F), scale bars = 100 nm, and the figures next to the micrographs show unit cells. Adapted from ref 76. Copyright 2017 American Chemical Society.

enzymatic activity of the catalase protein.74 This result shows that the CCM design rules that have been developed to control the crystallization of inorganic nanoparticles over the past two decades can be applied to control the structure of 3D protein materials. With these catalase building blocks, the flexibility of the linking unit between the oligonucleotide and the protein surface has also been found to be an important design parameter. When the flexibility of this portion of the building block was reduced, a transition to a crystal phase isostructural

Figure 5. Schemes of DNA design for (A) early 3D DNA-mediated protein assemblies in which complementary 20 bp sequences are conjugated directly to protein surfaces, and (B) using a linker-based design where recognition sequences (gray) are instead conjugated to protein surfaces. Linker strands (purple) are hybridized that are fully complementary to recognition sequences with an additional short single-stranded overhang, the sticky end, which mediates particle interactions and reorganization. 1943


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Accounts of Chemical Research with thorium phosphide was observed (Figure 6D−F). The decreased interprotein repulsion that occurs with the decreased conformational entropy in linkages with no PEG spacer was found to be the driving force for this change in thermodynamic structure.76 Protein Surface Chemical Topology Can Be Used To Control Crystal Structure

Protein surface chemical topology can be engineered to design directional interactions and therefore is an important design parameter in controlling the structure of protein superlattices. By control of the number and spatial position of DNA on the surface of a protein, the protein’s valency and therefore its crystallization behavior can be modulated. In the design of 1D assemblies, the number and position of DNA modifications were defined by genetic mutation. However, the spatial distribution of DNA can also be toggled simply by changing the chemical nature of DNA attachment to target different amino acid residues that naturally have different spatial distributions. To accomplish this objective, we used βGal again as a model system, as its lysine and cysteine residues are presented in differing numbers and positions on the protein surface, with approximately 8 cysteine residues located on the vertices of the protein and approximately 32 lysine residues distributed relatively evenly over the protein surface (Figure 7A). When βGal functionalized through its lysine residues was combined with complementary AuNPs, the expected CsCl lattice formed (Figure 7B). However, when the cysteine-functionalized protein was crystallized with AuNPs under otherwise identical conditions, an AB2 lattice formed as a result of the decreased number and specific positions of the DNA modifications accessed through the chemical topology of the protein (Figure 7C).77 Protein surface chemical topology can also be used to design and synthesize building blocks with multiple different types of oligonucleotides, for example, protein dimers with spatially defined arrangements of orthogonal DNA strands. Here, GFP containing a single cysteine surface mutation (described previously) was used to synthesize a conjugate that had a single DNA strand attached to its cysteine residue, and a different DNA sequence on its 18 surface lysine residues. When two GFP−DNA conjugates with complementary cysteine-appended strands were combined, a protein dimer formed in which different faces had different DNA sequences, hence forming a Janus protein particle (Figure 8A). This Janus protein, when crystallized with two sets of nanoparticles of either different sizes or compositions, each of which is complementary to a single face of the protein dimer, forms remarkable layered hexagonal architectures in which each of the DNA domains organized around a GFP dimer core interacts with a single layer of particles (Figure 8B−E).78 The ability to use protein surface chemistry along with multiple designed orthogonal DNA interactions to generate complex nanoscale building blocks such as the GFP-based Janus particle dramatically expands the scope of what is possible in colloidal crystal engineering with DNA.

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Figure 7. Chemistry of DNA attachment alters the crystallization outcome of proteins with complementary nanoparticles. (A) βGal can be modified through either its surface lysine or cysteine residues, which have different surface distributions. Crystallization with complementary AuNPs then yields body-centered cubic (BCC, CsCl) and AB2 lattices, respectively. (B) TEM micrograph of BCC crystals. Scale bars = 500 nm and (inset) 50 nm. (C) TEM micrograph of AB2 crystals. Scale bars = 1 μm and (inset) 50 nm. Adapted from ref 77. Copyright 2017 American Chemical Society.

interactions difficult to control also provides the ability to control valency through the known chemical topology of protein surfaces. The sequence and shape of the DNA and protein and the chemical nature of DNA attachment are all powerful design handles to manipulate the structure of 1D and 3D materials. Many exciting future opportunities and challenges remain in exploiting protein structural uniformity and tunable DNA placement to achieve unprecedented control over protein materials, ranging from highly ordered protein crystals to amorphous protein−polymer networks. A major challenge in designing other classes of materials, such as 2D crystals or 3D crystalline architectures with rotationally ordered proteins, remains the inherent flexibility of the linking unit between DNA and the protein surface, which may lead to many possible binding outcomes in the absence of a sufficient preference for directional assembly, as in the cases of GroEl and βGal (vide supra). A better thermodynamic understanding of how to design directional interactions on protein surfaces mediated by multivalent DNA binding needs to be developed to enable the rational design of these interactions. This will provide access to other classes of wellordered protein materials such as 2D crystals and 3D single crystals with rotationally ordered proteins. Growing protein single crystals through DNA interactions, where proteins are orientationally ordered such that angstromlevel structural information can be obtained, represents an

CHALLENGES AND FUTURE OUTLOOK

Protein materials engineering with DNA clearly offers routes to sophisticated 1D and 3D polymer structures as well as highly crystalline 3D architectures. A core idea driving this work has been that the same chemical heterogeneity that makes protein 1944


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even combine specific properties from multiple distinct proteins toward the synthesis of designer multivalent drugs or materials with applications in catalysis, separations, sensing, and protein structure determination. Finally, protein−DNA conjugates may allow many outstanding questions about the fundamental nature of DNAfunctionalized nanostructures to be answered. In contrast to other classes of DNA-functionalized particles, proteins enable the number and position of DNA to be discrete and programmable. Advances in cryo-EM techniques may enable the interaction of multivalent protein−DNA conjugates with membrane receptors to be elucidated. This knowledge would be revolutionary in the design and clinical translation of spherical nucleic acid (SNA) therapeutics.84−86 Furthermore, with proteins, local or global DNA density may be systematically varied to study the emergence of key SNA characteristics, such as cellular uptake, therapeutic activity, and cooperative binding. In the materials context, protein structural uniformity and DNA modification anisotropy may lead to novel nanoparticle phase behavior. Protein materials engineering with DNA offers a powerful platform to design tailored 1D polymers and 3D crystals. What we have learned from the control of structure in these systems will enable important material advances and fundamental studies in the future, including the synthesis of hydrogels with tunable mechanical properties and the development of new types of protein single crystals with applications in tissue engineering, catalysis, and protein structure determination. Overall, protein materials engineering with DNA promises unrivaled control over protein organization toward the creation and fundamental understanding of advanced materials.

Figure 8. Janus protein design and assembly. (A) Scheme of a Janus protein particle and its assembly with two sets of particles of differing composition (left) or size (right) with orthogonal DNA (purple and blue) on their surfaces. (B, C) Unit cells of layered hexagonal structures resulting from Janus protein crystallization. (D, E) TEM micrographs of the structure shown in (C). Scale bars = 150 nm. Adapted from ref 78. Copyright 2018 American Chemical Society.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ambitious unmet goal with far-reaching implications in both materials and structural biology. Current trial-and-error approaches to growing diffraction-quality protein crystals do not allow the crystal structure to be controlled, making protein structure determination a bottleneck in structural biology. Introducing programmable DNA interactions on protein surfaces may enable the structure of protein crystals to be engineered into highly porous or dynamic materials with applications in catalysis and sensing. Importantly, introducing DNA interactions on the surfaces of otherwise difficult-tocrystallize proteins may aid in inducing their crystallization through either DNA interactions alone or the DNA-mediated fusion of easily crystallized proteins, thereby leading to new strategies for protein structure determination. Using DNA to organize proteins offers a key advantage over other chemical strategies in that an almost infinite number of orthogonal interactions can easily be designed simply by altering the DNA sequence. Protein assemblies that take advantage of multiple orthogonal DNA interactions, however, require us to move beyond simple cysteine and lysine conjugation strategies and apply unnatural amino acid,79 Nterminus,80 and C-terminus81 conjugation strategies to these systems. With such additional chemical handles and the programmable orthogonality of nucleic acids, new structural complexity should be possible. Potential target structures include protein block, brush, and star polymers and amorphous protein networks with tunable mechanical properties82,83 and more complex crystalline morphologies. These materials may

ORCID

Janet R. McMillan: 0000-0002-3945-0194 Oliver G. Hayes: 0000-0002-9647-6411 Peter H. Winegar: 0000-0003-0984-4990 Chad A. Mirkin: 0000-0002-6634-7627 Notes

The authors declare no competing financial interest. Biographies Janet R. McMillan obtained her B.Sc. from McGill University (Montreal, Canada) in 2014 and completed her Ph.D. in Chemistry at Northwestern University in 2019, where she held a postgraduate fellowship from the National Science and Engineering Research Council of Canada. Her Ph.D. research focused on developing new strategies to organize proteins into well-defined architectures. Oliver G. Hayes is a Ph.D. candidate in Chemistry at Northwestern University. He received his M.Chem. in 2016 from the University of St Andrews (Fife, Scotland). His research interests include studying the design, synthesis, and properties of new protein−DNA materials. Peter H. Winegar received his B.S. in Chemistry from Michigan Technological University in 2017 and is currently pursuing a Ph.D. in Chemistry at Northwestern University. His research interests include the rational design of new classes of protein biomaterials. Chad A. Mirkin received his B.S. from Dickinson College in 1986 and his Ph.D. from The Pennsylvania State University in 1989. He was an 1945


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NSF Postdoctoral Fellow at MIT prior to becoming a professor at Northwestern University in 1991. He is the Director of the International Institute for Nanotechnology and the George B. Rathmann Professor of Chemistry, Chemical and Biological Engineering, Biomedical Engineering, Materials Science & Engineering, and Medicine at Northwestern University. He is an Associate Editor of JACS, a PNAS Board Member, and the founding editor of Small. He is a chemist and nanoscience expert who is known for his invention and development of spherical nucleic acids, dip-pen nanolithography, and related cantilever-free scanning-probe-based nanopatterning methodologies and his contributions to supramolecular chemistry and nanoparticle synthesis and assembly.

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ACKNOWLEDGMENTS The authors acknowledge support from the Vannevar Bush Faculty Fellowship Program, sponsored by the Basic Research Office of the Assistant Secretary of Defense for Research and Engineering and funded by the Office of Naval Research through Grant N00014-15-1-0043, and Air Force Office of Scientific Research Awards FA9550-16-1-0150 and FA955017-1-0348. J.R.M. gratefully acknowledges the National Science and Engineering Research Council of Canada for a postgraduate fellowship.

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Protein Materials Engineering with DNA | Accounts of Chemical

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