Supramolecular Protein Assemblies Based on DNA Templates

Aug 9, 2017 - Zhuhai United Laboratories Co., Ltd., Nation High & New Technology Industry Development Zone, Zhuhai 519040, China. ABSTRACT: DNA ...
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Supramolecular Protein Assemblies Based on DNA Templates Chunxi Hou,*,† Shuwen Guan,‡ Ruidi Wang,†,§ Wei Zhang,∥ Fanchao Meng,‡ Linlu Zhao,† Jiayun Xu,† and Junqiu Liu*,† †

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, and ‡College of Life Science, Jilin University, 2699 Qianjin Road, Changchun 130012, China § Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada ∥ Zhuhai United Laboratories Co., Ltd., Nation High & New Technology Industry Development Zone, Zhuhai 519040, China ABSTRACT: DNA plays an important role in the process of protein assembly. DNA viruses such as the M13 virus are typical examples in which single DNA genomes behave as templates to induce the assembly of multiple major coat protein (PVIII) monomers. Thus, the design of protein assemblies based on DNA templates attracts much interest in the construction of supramolecular structures and materials. With the development of DNA nanotechnology, precise 1D and 3D protein nanostructures have been designed and constructed by using DNA templates through DNA−protein interactions, protein−ligand interactions, and protein−adapter interactions. These DNA-templated protein assemblies show great potential in catalysis, medicine, light-responsive systems, drug delivery, and signal transduction. Herein, we review the progress on DNA-based protein nanostructures that possess sophisticated nanometer-sized structures with programmable shapes and stimuli-responsive parameters, and we present their great potential in the design of biomaterials and biodevices in the future.

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tracks, 2D planar DNA arrays/nanogrids, and 3D DNA lattices. Meanwhile, to construct more sophisticated DNA nanoarchitectures, DNA origami is preferred. DNA origami is a popular DNA nanotechnology in which a long ssDNA (general version is the M13mp18 plasmid) will assemble into programmed shapes in the presence of many “staple strands”.10 The discovery of DNA origami is a milestone in DNA nanotechnology, which profoundly impacts DNA research. Structural DNA nanotechnology provides a profuse amount of precise nanostructures that possess many functional groups and symmetrical sites for the design of protein assemblies, which could be functionalized by modifications with complementary ssDNA molecules, ligands, metal nanoparticles, and aptamers.11,12 Among these strategies, chemical modification allows DNA templates to be modified with functional groups of interest, such as biotin, amine, carboxyl, thiol, fluorescein, alkyne, and azobenzene.13,14 These modifications make DNA templates behave as desirable scaffolds to induce protein assemblies through protein−DNA interactions, DNA hybridization, and protein−ligand interactions. In the past decade, significant progress has been made in the field of DNA-based protein assemblies. Structural protein assemblies on DNA templates include 1D protein nanowires, 2D arrays of zinc finger proteins (ZFPs)/streptavidin (SA) proteins, and 3D nanohedra.15−18 A greater number of larger protein assemblies with ordered nanostructures could be

NA-templated protein assemblies, for example, the assembly of many coat proteins (CPs) on a genome, are naturally evolved, versatile systems with well-defined structures and functionalities.1,2 Among these assemblies, protein assemblies that possess catalytic, fluorescent, infectious, and receptor-binding abilities are interesting due to their potential in the next generation of optical/electronic and biomedical materials. Meanwhile, there are complex interactions in proteins including hydrogen bonding, electrostatic interactions, and hydrophobic interactions, which show limitations in the design of protein assemblies by protein− protein interactions.3−5 Comparably, it is facile to make DNA assemblies such as DNA walkers, DNA tweezers, and DNA rotors by predictable Watson−Crick base-pairing interactions, which result in distinct dimensional DNA nanostructures.6,7 These DNA nanostructures have been emerging as powerful templates for the exquisite design of protein assemblies. Structural DNA nanotechnology is successfully applied in the design of DNA nanostructures, including DNA tiles and DNA origami.8 DNA tiles are a class of assembly motifs in which several strands assemble into structural units such as triangular, rectangular, and cyclic shapes. Through hybridization of the protruding sticky ends, they will assemble with each other and extend to form periodical patterns in the plane and in space, resembling the assembly of protein building blocks in natural protein assemblies. Typical DNA tiles involve DNA helix bundles with cross-shaped junctions, such as double crossovers/triple crossovers (DX/TX crossovers), in which at least one single-strand (ss)DNA should span the neighboring DNA helix and the third DNA helix, respectively.9 As a consequence, they could form dimensional nanostructures such as 1D DNA © XXXX American Chemical Society

Received: June 21, 2017 Accepted: August 9, 2017 Published: August 9, 2017 3970

DOI: 10.1021/acs.jpclett.7b01564 J. Phys. Chem. Lett. 2017, 8, 3970−3979

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The Journal of Physical Chemistry Letters constructed by using enzyme-mediated approaches. For example, DNA tiles could assemble into ligase-mediated DNA crystals to encapsulate protein assemblies for biomedical and bioreactor applications. Herein, we review the recent progress in DNA-based strategies for protein assemblies, highlighting the potential in the design of antibody assemblies, enzyme assemblies, and drug delivery systems for multiple applications. Previous reviews on protein assembly and protein organization on DNA nanostructures and their applications are recommended for readers.19−22 DNA templates have been successfully applied in the design of 1D protein nanowires through protein−DNA interactions, the structures of which resemble those of natural architectures such as viruses, bacteria flagella, and microtubules.23−25 Through interactions with DNA templates, capping units, and metal nanoparticles, 1D length-controllable protein nanowires could be obtained, which offer a facile and flexible manner for the design, construction, and analysis of 1D protein structures. One-dimensional viruses provide good models to design 1D DNA-based protein assemblies because they contain only nucleotides (one to several DNAs or RNAs as the genome) and proteins.26,27 Mechanistic analysis indicates that the 1D viruslike nanostructure is induced by interactions of the stemlooplike origin of assembly (OAS) of the genome with coat proteins. Therefore, 1D virus-like assemblies could be induced by interactions of OAS-containing sequences and OAShybridized DNA with coat proteins. Interestingly, 1D viruslike structures could also be constructed from sphere-like nanostructures by using nonintrinsic DNA as templates. One-dimensional DNA-templated protein assemblies have been reported by Mukherjee and co-workers, in which coat proteins of a spherical virus (cowpea chlorotic mottlevirus, CCMV) assembled into nanotubes in the presence of nonspecific 500-mer ssDNA.28 The length of the DNAtemplated nanotubes was tunable by adjusting the base pair (bp)/CP ratio. Specifically, the lengths of the nanotubes followed a normal distribution, and nanotubes greater than 5 μm in length were formed at ratio of 5−10 bp/CP. Xu and coworkers have utilized shorter DNA fragments of 50, 100, and 300 bp heterogeneous DNAs as templates to induce the assembly of CPs of cucumber mosaic virus (CMV) into 1D protein nanotubes with a diameter of 17 nm (Figure 1a).29 The DNA-templated method is interesting because both natural CCMV and CMV are icosahedron viruses.31 A difference is that these DNA strands in both nanotubes have linear conformations instead of the coiled conformations of genome RNA and DNA in natural viruses.30 The Wege group has had many contributions regarding the DNA-based 1D bionic assembly of tobacco mosaic virus (TMV). They have made TMV assemblies by the hybridization of RNA to ssDNA templates on SiO2 substrates followed by RNAinduced TMV assembly (Figure 1b).32 Similarly, J. Eber et al. have used TMV-derived RNA to hybridize with the complementary oligonucleotides on gold nanoparticles, and after coassembly with TMV CPs, they constructed TMV nanotube-displaying gold nanoparticles with nanostar-like structures. This method generated nanostar-like gold nanoparticles with TMV nanotubes protruding with a high density.33 In order to endow the 1D virus with new functions, it is possible to modify the virus by point mutagenesis followed by site-directed bioconjugation. TMV nanotubes have been modified by diazonium coupling at tyrosine residues to give thiol groups, which could be used for the deposition of gold

Figure 1. Design of 1D protein assemblies on DNA templates. (a) Schematic of the 1D assembly of CPs induced by DNA templates. Adapted with permission from ref 29; Copyright (2008) the Royal Chemical Society. (b) Assembly of 1D tobacco mosaic virus (TMV)like nanostructures induced by OAS-ssDNA templates. Adapted with permission from ref 32; Copyright (2011) the American Chemical Society. (c) Process of the assembly of 1D amyloid fibrils onto DNA origami. Reprinted by permission from Macmillan Publishers Ltd.: Nat. Nanotechnol., ref 35, copyright (2014).

nanoparticles.34 Interestingly, they discovered that the full length of 300 nm in native TMV can be exceeded by using a longer RNA as the template. To create the longer RNA, the DNA with 8040 bp in length was amplified and transcribed so that it contained the TMV genome RNA linked with the partial sequence of the GUS gene before binding to CPs to coassemble. One-dimensional protein assemblies could also be constructed by the installation of a nucleation site on DNA origami. Udomprasert and co-workers have incorporated a nucleation site onto DNA templates for the design of 1D amyloid-like protein filaments. The nucleation site was successfully incorporated by linking the amyloid fibril peptide to a staple strand, which was located within tubular DNA origami (Figure 1c).35 Interestingly, when two tubular DNA origami were docked with one not containing a nucleation site, the amyloid fibrils could grow through both ends of the nanotubes. Due to the relationships between amyloid fibrils and diseases, the resulting amyloid fibrils are good models to analyze the cause of neurodegenerative diseases. The DNA-based strategies provide a powerful way to design 1D protein assemblies. It is possible to incorporate nicks, hairpin loops, ssDNA, DNA helices, DX, and TX into DNA nanostructures. The DNA-based protein assemblies not only have robust and periodic structures but also have functional 3971

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produce 2D protein patterns with exact and addressable structures. Metal−ligand interactions can be used to assemble proteins onto 2D DNA nanostructures. Meaningfully, the linkage between Ni-NTA and His-tagged protein could ensure sitespecific interactions. This is desirable for understanding protein assembly directed by DNA templates. Shen et al. have conjugated thio-modified DNA strands to NTA by maleimido chemistry. Subsequently, the origami composed of maleimidoNTA/DNA, DNA staple strands, and a single-stranded M13mp18 DNA plasmid has been used to generate a 2D pattern of EGFP proteins and for deposition on a poly-L-lysine pretreated mica surface (Figure 3a).46 In the same way, Goodman and co-workers found that clustering NTAcontaining ssDNA with three NTA molecules on its end could induce the formation of a protein−His6:Ni2+:trisNTADNA complex and result in a retention shift in liquid chromatography after Ni2+ chelation, while single NTAcontaining ssDNA could not.47 By making use of this method, they have constructed 2D green fluorescent protein (GFP) arrays on 2D planar kagome, which was composed of four different ssDNA, and fluorescence microscopy confirmed that His6-tagged GFP could assemble into internal kagomes. These approaches require chemical modifications and hybridizations to fabricate protein assemblies on DNA templates, inevitably leading to incomplete valent protein assemblies. Meanwhile, the incorporation of special DNA sequences into DNA templates can address the challenge, for example, aptamers and ZFP-binding motifs have been designed and incorporated into DNA scaffolds. Aptamer−protein interactions have been used for the design of 2D protein assemblies. Chhabra and co-workers have designed four DX crossover DNA, namely, A tile, B tile, C tile, and D tile, to construct a 2D DNA sheet (Figure 3b).48 When the B tile and D tile were modified with respective aptamers, the 2D DNA nanostructures could be used for the assembly of patterned thrombin and platelet-derived growth factor (PDGF) on periodic 2D DNA nanoarrays that contained linear-shaped or “S”-shaped human α-thrombin-binding aptamers and PDGF-binding aptamers. DNA-binding proteins are good models for 2D assembly on DNA scaffolds such as ZFPs, which could undergo a rapid and reversible assembly process. First, ZFPs of zif268 and AZP4, capable of binding to 10 bp of DNA specifically, have been successfully assembled on the rectangular DNA origami (Figure 3c).49 Significantly, the binding efficiency of AV-Zif268 at the central cavity increased more than 70%, and the equilibrium dissociation constant was 63 nM, enabling efficient assembly of multiple proteins. Similarly, the site-directed and reversible assembly of ssDNA-binding proteins on DNA resins has been achieved, which could be disassociated by changing the pH, divalent cations, and complementary oligonucleotides.50,51 These protein assemblies pave the way for protein purification and biosensors. Generally, 3D protein nanostructures are the essential components of natural cells, organelles, supporting structures, and microorganisms.52,53 To address the challenge in microscopic design, DNA nanotechnology has evolved a large number of 3D DNA nanostructures with nanoscale resolution. These 3D DNA nanostructures can be used for the construction of 3D protein nanostructures. The resulting protein assemblies possess exposed binding sites for catalytic

groups and entities on the outer surface. Thus, proteins could coassemble with metal nanoparticles, polymers, and other materials into a variety of versatile protein assemblies.36 These complexes behave as smart biomaterials for applications in catalysis, fluorescence, and drug delivery. There is great progress in the design of 2D protein assemblies on DNA nanostructures. A general method is to convert 2D DNA arrays to 2D protein arrays by modifications of DNA templates at specific sites.37−39 The modification of DNA templates with ligands such as biotin, nitrilotriacetic acid (NTA), and aptamer enables protein−ligand interactions, Histag−metal interactions, and protein−aptamer interactions for protein assembly.40−42 These methods have many advantages because they could be used for the assembly of a variety of protein assemblies with identical orientations. Biotin−SA interactions have been used for 2D protein assembly on DNA templates. Numajiri and co-workers have designed a U-shaped nine-helix bundle with two junctions in the interiors of every two neighboring helices for fixation. When two U-shaped bundles were linked in the presence of a tribiotinylated strand, a tribiotin-containing well in rectangular DNA origami was formed, enabling the unsaturated binding of SA tetramers (Figure 2a).43 Thus, 2D DNA nanostructures

Figure 2. DNA-templated 2D protein assemblies. (a) Schematic drawing of the 2D self-assembly of SA on well-containing rectangular DNA origami. Adapted with permission from ref 43; Copyright (2010) the American Chemical Society. (b) 2D protein arrays templated by 4 × 4 DNA nanogrids. Adapted with permission from ref 44; Copyright (2003) AAAS. (c) TX crossover DNA-based 2D SA assemblies. Adapted with permission from ref 45; Copyright (2004) the American Chemical Society.

were facile to convert to 2D protein nanogrids through biotin− SA interactions. Yan et al. designed a four-arm DNA tile that consisted of nine strands and four bulged T4 loops, the latter of which took part in binding four arms and forming specific angles between the arms. The DNA tiles assembled into 4 × 4 tiles, 2D nanogrids step by step, and 2D protein nanogrids in the presence of SA (Figure 2b).44 Similarly, Li and co-workers designed TX crossover DNA-based nanolines. The TX crossover DNA contains one biotinylated loop on the upper helix and one biotinylated loop on the lower helix, enabling the assembly of SA proteins alternatively on both sides of nanolines (Figure 2c).45 These approaches provide a convenient way to 3972

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Figure 3. DNA-templated 2D protein assemblies. (a) Schematic of Ni-NTA-DNA-directed 2D protein assemblies. Adapted with permission from ref 46; Copyright (2009) the American Chemical Society. (b) Schematics of 2D DNA nanoarrays with alternate thrombin- and PDGF-binding aptamers and assemblies of their target proteins. Adapted with permission from ref 48; Copyright (2007) the American Chemical Society. (c) 2D assemblies of ZFPs induced by DNA origami. Adapted with permission from ref 49; Copyright (2012) Wiley.

functions and synergistic effects, making them useful in catalysis, sensors, and medicine. 3D protein assemblies have been assembled on DNA templates by the modification of DNA templates with ligands, followed by ligand-mediated assembly. Three-dimensional assemblies of SA proteins onto each face of a 3D DNA tetrahedron (TET), octahedron (OCT), and icosahedron (ICO) have been designed and produced. For example, Zhang and co-workers have scrutinized such a two-step procedure to align the SA proteins onto the facets of 3D nanohedra by using SA−biotin interactions (Figure 4a).54 They designed three DNA strands that contained large repeated strands capable of forming star-shaped DNA motifs as well as medium strands and biotin-conjugated short strands to assemble into polyhedra. Mixing of the SA and DNA nanohedra allowed the formation of trivalent binding, resulting in 3D protein assemblies. These 3D protein assemblies possessed unsaturated binding and stronger affinity than monovalent binding. It should be noted that the icosahedron structure with well-defined symmetric nucleic acids and proteins resembles that of an icosahedron virus, which lays a foundation for the study of self-assembly mechanisms of viruses and biomedical applications such as multivalent vaccines. Furthermore, they have investigated the chirality of proteins on DNA octahedra.55 Structure analysis indicates that the tile would bend inward rather than in the outer direction because when the DNA tile is rotated by 90° there is more space in the back face than in the front face. The above strategies show the potential in the design of bionic protein assemblies, some of which have similar high-order nanostructures to those of natural protein assemblies. 3D protein assemblies have also been designed by orthogonal strategies, such as a combination of protein−DNA conjugation and DNA assembly.56,57 In terms of this orthogonal strategy, click chemistry is a popular method because of its high efficiency and specificity. Duchworth et al. have synthesized alkyne-modified DNA that could assemble into a DNA tetrahedron and then reacted it with azide-containing GFP by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition reaction.56 In this case, to synthesize azide-containing GFP, GFP was genetically fused with a C-terminal tetrapeptide CVIA, which was modified by farnesyltransferase (PFTase) to incorporate azide-modified isoprenoiddiphosphate (Figure 4b). This study

Figure 4. DNA-templated 3D protein assemblies. (a) 3D assembly of proteins onto each surface of a 3D DNA tetrahedron. Adapted with permission from ref 54; Copyright (2012) Wiley. (b) Design of 3D protein assemblies by using transferase-modified DNA nanostructures. Adapted with permission from ref 56; Copyright (2007) Wiley.

indicates that orthogonal approaches are ideal strategies for the induction of 3D protein assemblies. 3973

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The Journal of Physical Chemistry Letters Other orthogonal approaches include self-labeling fusionprotein techniques, including Snap-tag, haloalkane-dehalogenase (Halo-tag), and O6-alkylguanine-DNA-alkyltransferase (hAGT).58 Meyer and Niemeyer have efficiently coalesced a self-labeling protein technique (Snap-tag and Halo-tag), biotin−SA interactions, and antigen−antibody interactions to modify a four-way DNA motif.59,60 However, a shortcoming of this system is the possible steric hindrance, which may cause low efficiencies in Snap-mediated and Halo-tag-mediated coupling with DNA templates. In the cellular context, all events happen in high concentrations of cytosol and compartments. The high concentrations of context are not desirable for consequent reactions. To enable metabolic flux, it is preferred to assemble multiple enzymes into large assemblies such as enzyme complexes of proteasomes as well as photosynthetic system and respiratory chain supercomplexes. Enzyme assembly makes it possible to locate many active centers in close proximity in space, thus improving the local concentrations of reactants and intermediates.61 As a result, enzyme assembly could make use of multiple enzymes cooperatively and synergistically.62 DNA-templated multiple enzyme complexes (MEC) can realize the cascade reaction, regeneration of cofactors, and Förster resonance energy transfer (FRET) by adjusting the distances between enzymes on DNA templates. Thus far, DNAtemplated MEC have been reported, which could be used for chemical analysis and detection. Niemever et al. have reported in vivo biotinylated luciferase (Luc) and NAD(P)H:FMN oxidoreductase (NFOR) and their assembly on ssDNA-SA templates, followed by assembly on double-strand DNA templates for cascade reactions.63 They found that steric hindrance affected the signal intensities more than the sequence-specific binding. Meanwhile, cascade reactions that happen on the inside of a nanoreactor are preferred to mimic natural reactions in the cell interior. Linko et al. have anchored GOx or HRP within disk-like origami by biotin−neutravidin interactions and glued them along a symmetrical axis into tubelike nanoreactors (Figure 5a).64 For a further increase of the cascade efficiency, the incorporation of cofactors between enzymes is desirable. Fu et al. have described such a method by introducing a swing arm of NAD+-poly(T) between glucose-6-phosphate dehydrogenase (G6PD) and malic dehydrogenase (MDH) on DNA nanolines.65 Competitive experiments showed that the G6pDHNAD+-MDH on a DNA nanoline displayed a channel for NADPH to transfer from G6pDH to MDH, efficiently avoiding the competition from LDH in solution (Figure 5b). The success encourages attempt to design a switchable cascade system. Azobenzene is a light-responsive molecule that has been introduced into a DNA strand to switch the glucose oxidase (GOx)/horseradish peroxide (HRP) system.66,67 UV light allows azobenzene to follow a trans-to-cis conversion, decreasing the binding of a DNA duplex to keep the enzymes separated. Visible light irradiation induces the cis-to-trans conversion, thus promoting the cooperative activities (Figure 5c). Antibody drugs account for a large portion of the drug market. Traditionally, these antibody drugs have been produced by antibody fusion through genetic engineering and antibody modification with target molecules, PEG, and other molecules to enhance the efficiency, stability, and in vivo circulation.68−70 Meanwhile, future medical therapy requires precise organization of multiple antibodies and integration of the antibodies

Figure 5. DNA-templated protein assemblies for cascade reactions. (a) CanDo-simulated two DNA origami nanoreactor for cascade reactions. Adapted with permission from ref 64; Copyright (2015) the Royal Society of Chemical. (b) Design of a NAD+-modified swinging arm for the restricted diffusion of NAD+/NADH between two dehydrogenases on a DNA nanoline. Reprinted by permission from Macmillan Publishers Ltd.: Nat. Nanotechnol., ref 65, copyright (2014). (c) Lightresponsive azobenzene-integrated DNA duplex that controls cascade activities of glucose oxidase (GOx)/horseradish peroxidase (HRP). Adapted with permission from ref 66; Copyright (2011) the American Chemical Society.

into miniaturized devices. For this aim, DNA-templated antibody assemblies in solution and on surfaces have been designed and produced. Antibody assemblies have been recently developed based on DNA templates, which are especially useful in diagnostic and therapeutic applications. He et al. designed nine single strands, which could form 2D DNA arrays with repeating distances of 19 nm. Antibodies can be assembled on this DNA scaffold after covalent modification of the central DNA strand with antigen fluorescein (Figure 6a).71 These antibodies have similar orientations and high densities, providing a good material for biomedical analysis and practical applications. 3974

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the gold surface, which became a multichannel biosensor with high specificity toward the respective antigens. DNA templates could be used to assemble proteins for the development of drug delivery systems. An interesting area of research is to switch the DNA templates off to release payloads by stimuli such as pH, redox, metal ions, light, heat, and aptamers.9,21,22,74 Recently, this has been undoubtedly attractive for aptamers because different cells have distinct responses to aptamers so that aptamer-based drug delivery systems could release drugs dependent on cells. Douglas and co-workers have designed an aptamerresponsive DNA nanorobot for in vivo research areas such as cell targeting, payload transporting, and signaling activation (Figure 7a).75 The logical and gated barrel-like nanorobot was composed of two domains, which were linked by singlestranded scaffold hinges and fastened by DNA aptamerattached staples. Different cell lines expressed different levels of “key” proteins that bind to the respective aptamers. For example, a neuroblastoma cell line can activate nanorobots with two sgc8c locks. Furthermore, it can be unpicked to expose the

Figure 6. DNA-templated antibody assemblies in solution and on surfaces for applications. (a) Scheme of the DNA-templated selfassembly of antibody (IgG) arrays. Adapted with permission from ref 71; Copyright (2006) the American Chemical Society. (b) Scheme of the synthesis of the Fab′−DNA conjugate and antibody assembly by hybridization. Adapted with permission from ref 72; Copyright (2013) the American Chemical Society. (c) Schematic of DNA-templated assemblies of DNA−antibody conjugates on a gold surface. Adapted with permission from ref 73; Copyright (2006) the American Chemical Society.

DNA templates can mediate antibodies to assemble onto a profuse number of surfaces by chemical modifications. Coyle et al. have reported the DNA-directed assembly of antibody heterodimers on a membrane surface (Figure 6b).72 In this study, a strand of ssDNA was functionalized with thiols, supported membranes were modified with maleimide, and Fab′ fragments were obtained by treatment of polyclonal donkey antimouse antibodies with 2-mercaptoethylamine, followed by modification with complementary ssDNA. Once the soft surface is substituted by a solid surface for protein assembly, DNA template−protein assemblies can be used for biosensors. Boozer et al. have described a DNA-based method for the detection of multiple fertility hormones (Figure 6c).73 They modified antibodies with DNA by reacting them with sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate and thiolated ssDNA sequentially. Through DNA hybridization with complementary ssDNA, the antibodies were assembled on

Figure 7. DNA-templated 2D protein assemblies. (a) Design of an aptamer-gated DNA nanorobot for drug delivery. Adapted with permission from ref 75; Copyright (2012) AAAS. (b) Schematic diagram of DNA hydrogels mediated by T4 DNA ligase for drug delivery. Reprinted by permission from Macmillan Publishers Ltd.: Nat. Mater., ref 76, copyright (2006). 3975

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The Journal of Physical Chemistry Letters stapled antibody payloads for cell surface recognition and signal activation. Nanorobots were modified with 41t locks to target NKL cells, and the two encapsulated antibodies against human CD33 and against human CDw328Fab′ could induce growth arrest and inhibit the Jun N-terminal kinase and Akt (protein kinase B) signals. DNA could also be used to construct hydrogels to encapsulate active proteins for drug delivery. A facile way is to convert the nanostructures of 2D DNA arrays to hydrogels and other macroscopic structures by ligase. These hydrogels maintained the structural and physicochemical properties of DNA tiles. Interestingly, changing one of the primary sequences of the DNA motifs may lead to changes in the structural, physicochemical, and mechanical properties of DNA hydrogels. Um and co-workers have described landmark work on DNA hydrogels. These DNA-based hydrogels were formed by the enzyme-catalyzed assembly of branched X-DNA, Y-DNA, and T-DNA molecules (Figure 7b).76 In addition to the initial concentrations, the strategy provides a facile way to regulate the trends of the swelling of hydrogels by changing the branched DNA types. This research makes it possible to assemble functional biomaterials programmable from the nanoscale to macroscale. DNA-templated assembled proteins have been successfully used for in vivo studies.77,78 Because the distance and number of receptor-binding proteins on DNA can be adjusted, these DNA−protein assemblies may activate many signal transductions such as receptor phosphorylation, endocytosis, and apoptosis pathways.8,19,20 Fujita and co-workers have designed an equilateral-triangular RNA−protein complex to activate apoptosis by inducing the clustering of cell surface receptors (Figure 8a).79 The fusion proteins of L7Ae-Gβ1x2 capable of sequence-specific binding at the apexes could significantly improve adherence to the cell surface receptors. Their research showed that signal activations may depend on the sizes of Tri-RNAs. With respect to the five Tri-RNAs with 15, 26, 48, 70, and 92 bp by length, Tri-RNP15Gal1β promoted apoptosis the most because it caused closely stacked lattice-like nanostructures, which could induce the assembly of glycoreceptors to activate signal transduction for the apoptosis of T cells. Shaw and co-workers have designed 18 parallel double helixcomposed nanotubes with two pairs of protruding ssDNA handles along the tube axis, which were separated by 42.9 and 100.1 nm. The binding sites allowed them to bind protein− ssDNA conjugates of Ephrin-A5-Fc-ssDNA, forming nanocalipers (NC40 and NC100) (Figure 8b).80 Interestingly, NC40 increased receptor phosphorylation more than NC100, although they showed similar higher binding affinities than that of NC0. The above results indicate that the proximity is the dominant impact factor other than binding affinity in EphA2 receptor phosphorylation. In the past decade, the field of protein assemblies induced by DNA templates has undergone rapid progress. These successes could be ascribed to understanding of natural protein−DNA complexes and development of DNA nanotechnology. Viruslike assemblies have been successfully constructed based on DNA templates, which could shed light on the detailed assembly mechanism of natural protein assemblies. Alternatively, to address the challenge in the design of sophisticated protein assemblies on the nanoscale, DNA nanotechnologies have been well-developed, such as DNA tiles, DX/TX

Figure 8. Applications of DNA-templated protein assemblies in vivo. (a) Illustration of apoptosis regulation by manipulating the distances between the receptor components with small and large Tri-RNPs. Reprinted by permission from Macmillan Publishers Ltd.: Sci. Rep., ref 79, copyright (2014). (b) Phosphorylated EphA2 receptor (purple dots) in MDA-MB-231 cells cultured on fibronectin micropatterns and detected by PLA. Reprinted by permission from Macmillan Publishers Ltd.: Nat. Methods, ref 80, copyright (2014).

crossovers, and DNA origami. Generally, DNA tiles could make use of a short number of designed DNA, while DNA origami needs a large number of designed ssDNA to extend to dimensional structures in the plane and in space. These technological breakthroughs make it possible to produce highorder protein assemblies on the nanoscale. DNA nanotechnology has been used to design dimensional protein assemblies and functional protein assemblies for cascade reactions, biomedicine, and signal transduction. It is worth noting that the recreation of receptor-binding protein assemblies is essential for biophysical analysis and signal transduction applications. Signal transductions can be enhanced by changing parameters such as the distance, position, and orientation of individual proteins on DNA templates. By changing these characteristics, it is possible to design efficient DNA-templated assemblies to activate special signals, such as receptor phosphorylation, endocytosis, and apoptosis. In recent progress, more articles have focused on the construction of homogeneous protein assemblies (dimensional protein nanostructures and crystals) on DNA templates and their in vitro applications. In contrast, nature exerts sophisticated functions and often uses heterogeneous protein assemblies such as proteasome and hybrid protein assemblies including ferritin (a multimeric iron storage protein with ferroxidase activity, 12 copies in prokaryotic cells, 24 copies in eukaryotic cells). To construct heterotropic protein assemblies, it is possible to apply distinct DNA-binding proteins, aptamerbinding proteins, and ssDNA−proteins to DNA templates. Heterotropic assembly could also be achieved by postassembly 3976

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Letter

The Journal of Physical Chemistry Letters

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modifications. Besides in vitro applications, in vivo applications of protein assemblies including receptor-binding proteins and antigens have been developed. In the future, more DNA-based protein assemblies will be developed for biomedical applications. Specifically, short peptide motifs such as RGD (a canonical ligand of integrin receptor αvβ3) could be integrated into protein assemblies for transmembrane transport and drug delivery systems, and for in vivo intranuclear applications, several hormones could be introduced in protein assemblies for translocation into the nucleus. We envision that there will be an encouraging development in the design of protein assemblies based on DNA templates in the future. Meanwhile, there is limited research on their dynamics and kinetics on cascade reactions, diagnostics, therapies, and signal transductions. These studies could provide useful information to incorporate feedback or feed-toward designs for biomedical applications. For example, Liu et al. have found that there was a kinetic bottleneck from the closed state to open state of DNA tweezers in the enzyme nanoreactor. Therefore, in order to switch the open/closed states quickly in the future, the reaction could be accelerated for a set strand with a self-folded hairpin in the closed tweezers.81 Taking these into consideration, DNA motors, dynamic rotors, machines, and factories could be used for exquisite protein assemblies, which lays a foundation for precise diagnostics and therapy.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.H.). *E-mail: [email protected] (J.L.). ORCID

Junqiu Liu: 0000-0002-8922-454X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge support from the Natural Science Foundation of China (No: 21234004, 21420102007, 21574056, 91527302) and the Chang Jiang Scholars Program of China.

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REFERENCES

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