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Peptide-oligonucleotide hybrid molecules for bioactive nanomaterials Nicholas Stephanopoulos Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00259 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Bioconjugate Chemistry

Peptide-oligonucleotide hybrid molecules for bioactive nanomaterials Nicholas Stephanopoulos1,2 1. School of Molecular Sciences; 2. Biodesign Center for Molecular Design and Biomimetics Arizona State University, Tempe AZ 85251 ([email protected]) Abstract: Peptides and oligonucleotides are two of the most interesting molecular platforms for making bioactive materials. Peptides provide bioactivity that can mimic those of proteins, whereas oligonucleotides like DNA can be used as scaffolds to immobilize other materials with nanoscale precision. In this Topical Review, we discuss covalent conjugates of peptides and DNA for creating bioactive materials that can interface with cells. In particular, we focus on two areas. The first is multivalent presentation of peptides on a DNA scaffold, both linear assemblies and more complex nanostructures. The second is the reversible tuning of the extracellular environment— like ligand presentation, stiffness, and hierarchical morphology—in peptide-DNA biomaterials. These examples highlight the potential for creating highly potent materials with benefits not possible with either molecule alone, and we outline a number of future directions and applications for peptide-DNA conjugates. Introduction. In nature, molecules like proteins, lipids, and DNA control the self-assembly, structural properties, and nanoscale presentation of bioactive signals. Reproducing these properties in synthetic materials is critical for interfacing them with biological systems, for applications that include: therapeutic cargo delivery, target sensing and imaging, tissue engineering, and fundamental studies of biological mechanisms and forces. In recent years, two molecular platforms that have found increasing use in constructing self-assembling materials are DNA and synthetic peptides. DNA—with the highly predictable Watson-Crick base pairing rules— provides almost limitless programmability for the synthesis of complex nanostructures,1 sequence-specific immobilization of multiple ligands, and reversible linking of two components through strand-displacement reactions.2 Peptides, by contrast, often reproduce biological effects of full-length proteins, but have the advantage of being shorter and more synthetically tractable via solid-phase synthesis, which enables the incorporation of non-canonical synthetic amino acids. Peptides also possess a rich self-assembly behavior, which can generate nanostructures as extracellular matrix (ECM) mimetic scaffolds for tissue engineering.3 In recent years, covalent peptide-DNA hybrid molecules have enabled a range of materials that merge the programmability and key functional properties of DNA nanotechnology with the bioactivity and structural and chemical diversity of peptides. In this Topical Review, we will discuss peptide-DNA nanomaterials in two emerging areas that show particular promise for creating bio-functional materials. We will first discuss using DNA as a programmable scaffold to control the nanoscale spacing of multiple peptides, in order to enhance target binding compared with monomeric peptides (Figure 1A,B). Second, we will describe the synthesis and application of peptide-DNA biomaterials to tune the extracellular environment, primarily to reversibly control the ligand presentation, stiffness, and hierarchical morphology of biomaterial scaffolds (Figure 1C,D). Peptide-oligonucleotide conjugates that fall outside the narrow purview mentioned above will not be covered here, and we refer the interested reader to reviews on the many additional rich applications of these materials.4, 5 We will also restrict the discussion to covalent bioconjugates of synthetic peptides and DNA (or in some cases analogues, like peptide nucleic acid (PNA)), as opposed to noncovalent association of the two components,6-8 or materials that use recombinant proteins.9 However, even within this scope, we hope to convey the great potential for peptide-DNA

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conjugates for building highly biomimetic systems that can probe fundamental biology and enable applications in targeted therapies and regenerative medicine. The peptide-DNA conjugates described herein were all synthesized via chemical coupling of modified oligonucleotides and synthetic peptides, either directly or through the use of bifunctional crosslinkers (Figure 1E). However, several reports below utilized PNA-peptide hybrids, which can be synthesized as a continuous molecule on solid-phase support (Figure 1F). For a more thorough treatment of peptide-oligonucleotide bioconjugation strategies, we refer the reader to more in-depth reviews.4,5

Figure 1: Peptide-DNA nanomaterials. A) DNA-peptide conjugates can be used to tune the distance between the peptides using a complementary strand linker. B) Self-assembly of DNA nanostructures (e.g. a tetrahedral cage) from DNA-peptide conjugates and unmodified strands. C) Toehold-mediated strand displacement to remove a bioactive peptide from a biomaterial surface. D) Reversible crosslinking of peptide-DNA hydrogels to tune mechanical properties and hierarchical assembly. E) Synthesis of DNA-peptide conjugates using a bifunctional crosslinker (like the amine- and thiol-reactive linker SPDP). F) Synthesis of PNA-peptide conjugates through sequential on-resin coupling of PNA monomers followed by amino acid monomers. Multivalent peptide display on DNA scaffolds. Although short peptides can be more stable and scalable than full-length, expressed proteins, they are often less active than the molecules from which they are derived. One strategy to enhance their potency is to create multivalent assemblies of identical peptides, or co-localize two different peptides with synergistic effect. DNA is a natural choice for linking them into such assemblies because its programmable sequence and monodisperse length allow a stoichiometrically-defined number of different molecules to be integrated through peptide-DNA hybrids bearing a complementary sequence. This property is in


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contrast to other self-assembling systems (e.g. peptide nanofibers), which rely on less specific supramolecular forces, and generally yield a highly symmetric, repeating assembly of epitopes as opposed to a discrete monomeric assembly. In 2009, the Johnson and Chaput labs demonstrated that DNA could be used to link two low-affinity peptides into a much higher affinity agent, which they termed a “synbody” (for “synthetic antibody”).10 Two micromolar-affinity peptides—themselves derived from a library of random peptide sequences synthesized on a chip—were linked to DNA handles using a heterobifunctional linker. The two molecules could then be co-localized in space using a DNA strand complementary to both unique handles (Figure 2A). This DNA linker could tune both the distance between the peptides, but also the angle between them due to the helical nature of double-stranded DNA (Figure 2B). The optimal synbody resulted in a ligand with 5 nM affinity to the yeast regulatory protein Gal80, an increase of three orders of magnitude over the monomeric peptides, by binding to two different faces of the protein. This “clamp”-like behavior is highly mimetic of natural binding agents, like antibodies, but without relying on a complex (and often intractable) protein scaffold to position the key binding residues. Shortly after the work highlighted above, a number of reports demonstrated the great power of multivalent peptide assemblies on DNA scaffolds. In 2010, the Winssinger laboratory demonstrated that cyclic peptide inhibitors of the TRAIL death receptor 5 (DR5) could be dimerized using a DNA linker11 The peptides were synthesized with peptide nucleic acid (PNA) handles, an uncharged oligonucleotide that binds via Watson-Crick pairing to DNA, and the distance between them was tuned using both PEG spacers and the DNA scaffold (Figure 2C). The optimal combination of linkers and distance between ligands led to a 5-fold increase in affinity over the monomeric ligands, whereas non-optimal presentation of the signals gave affinities similar to (or even worse than) the monomer. However, these combinations could be screened quickly and in parallel using different DNA scaffolds, obviating the need for laborious synthesis of covalent dimers. In 2011, the Seitz group also used DNA in this fashion, as a “molecular ruler”, to probe the distance dependence for dimeric binding of a phosphopeptide to the Syk-tSH2 kinase domain (Figure 2D)12 The exact distance between ligands could be tuned by the number of intervening nucleotides in the DNA linker, and the flexibility tuned by its single- vs. doublestranded nature, or the introduction of nick points. The Seitz lab followed up this work with two additional reports that used DNA linkers to probe bivalent peptide binding to the adaptor complex 2 (AP-2)13 or peptides that could distinguish between Zap-70 and Syk tSH2 binding.14 In this second report, the results from screening templated peptides on DNA led to a distance dependence that could be reproduced with a non-oligonucleotide linker, demonstrating the advantages of DNA for rapid screening followed by translation to a more scalable molecular platform. The bivalent nature of heterodimeric peptide conjugates was used by Merkx and colleagues to create high-affinity blocking agents for antibodies.15 By linking the antibody-binding peptide to the DNA linker via a protease-cleavable linker, the divalent blocking group could be cleaved in the presence of the enzyme MMP2, activating the antibody in the tumor microenvironment.



Figure 2: Scaffolding multiple peptides and PNA-peptide hybrids on linear DNA. A) Design of a “synthetic antibody” that uses a DNA linker to co-localize two peptides that bind to different interfaces of a target protein. B) Tuning the distance and angular relationship between the two peptides using the DNA linker. Reprinted (Adapted or Reprinted in part) with permission from Williams, B. A. R., Diehnelt, C. W., Belcher, P., Greving, M., Woodbury, N. W., Johnston, S. A., Chaput, J. C. (2009) Creating Protein Affinity Reagents by Combining Peptide Ligands on Synthetic DNA Scaffolds. J. Am. Chem. Soc. 131, 17233-17241. Copyright 2009 American Chemical Society. C) Peptide-PNA hybrids can be used to generate a dimer that mimics a multivalent protein binding to its receptor. Reprinted (Adapted or Reprinted in part) with permission from Gorska, K., Beyrath, J., Fournel, S., Guichard, G., Winssinger, N. (2010) Ligand dimerization programmed by hybridization to study multimeric ligand-receptor interactions. Chem. Commun. 46, 7742-7744. Copyright 2010 Royal Society of Chemistry. D) DNA linkers can be used as a “molecular ruler” to change the distance and flexibility between signals. Reprinted (Adapted or Reprinted in part) with permission from Eberhard, H., Diezmann, F., Seitz, O. (2011) DNA as a Molecular Ruler: Interrogation of a Tandem SH2 Domain with Self-Assembled, Bivalent DNA-Peptide Complexes. Angew.Chem. Int. Ed. 50, 4146-4150. Copyright 2011 John Wiley and Sons. E) Generation of highly multivalent PNA-peptide hybrids by immobilization to a DNA scaffold with repeating complementary sequences. Reprinted (Adapted or Reprinted in part) with permission from Englund, E. A., Wang, D. Y., Fujigaki, H., Sakai, H., Micklitsch, C. M., Ghirlando, R., Martin-Manso, G., Pendrak, M. L., Roberts, D. D., Durell, S. R., Appella, D. H. (2012) Programmable multivalent display of receptor ligands using peptide nucleic acid nanoscaffolds. Nat. Commun. 3, 614. Copyright 2012 Springer Nature. The above examples all created either homo- or hetero-bivalent peptide assemblies to bind to two distinct entities. An alternative approach was reported by the Appella lab in 2012, whereby a DNA scaffold was used to create a multivalent presentation of up to 45 binding peptide-PNA conjugates (Figure 2E).16 In this fashion, the number of ligands (the integrin-binding cyclic RGDfK


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peptide) could be tuned simply by changing the DNA scaffold (which is straightforward) rather than synthesizing a highly repetitive covalent polymer (which is more difficult). Over 52 systems were screened for inhibition of melanoma cells (which overexpress the target integrins), and the construct with 15 peptides was found to maximize efficiency; above that number no increase in activity was seen, likely due to spatial saturation of the receptor. A natural extension of this system would be to immobilize multiple different peptides, with stoichiometric and nanoscale control (as demonstrated by Stupp and coworkers, vide infra). In this fashion, the complex extracellular milieu seen by cells, with a large number of protein ligands arrayed in precise distance relationships, could be reproduced quickly. All the reports mentioned thus far used a relatively simple, linear DNA scaffold to display multiple peptides. However, in the past 30 years the field of DNA nanotechnology has developed complex structures from oligonucleotides, thanks to the large number of orthogonal interactions possible through Watson-Crick pairing. These include two- and three-dimensional objects (e.g. DNA origami,1, 17, 18 single-stranded tiles/bricks19-21) with a degree of addressability and anisotropy not possible with other molecular platforms. By attaching peptides to the constituent strands that make up these self-assembled nano-objects, it is possible to control the spacing and valence of these signals in a way not possible with a simple linear arrangement on a DNA duplex. In 2017, Sacca and coworkers demonstrated that a DNA nanostructure bearing multiple copies of a binding peptide could be used to “wrap” an oligomeric protein through many, spatially-matched peptide-protein interactions (Figure 3A).22 The authors targeted the serine protease DegP as the target, a protein that can exist in multiple oligomeric states ranging from 6 to 24 monomers. A short peptide (DPMFKLV) that targeted the protein was conjugated to a DNA handle and immobilized on the inside of a hexagonal DNA origami barrel 20 nm across, a good size match for the 24-mer but not the smaller oligomers. Up to 18 peptides could be attached to the origami, allowing it wrap around the desired target—efficiently and at a 1:1 stoichiometry—while excluding the smaller assemblies. In many ways, this work was similar in principle to the synbody approach,10 but extended to a much larger structure: the DNA scaffold effectively recapitulates a protein-protein interface that would be very difficult, if not impossible, to achieve with native proteins. This approach also has the advantage of effectively blocking the interactions of the target protein with any other molecules, either for purification from a mixture, or potentially to disable a pathogenic protein.



Figure 3: Multivalent peptide display on DNA nanostructures. A) Tethering a binding peptide to the inside of a DNA origami barrel allows for multivalent “wrapping” of the target protein. Reprinted (Adapted or Reprinted in part) with permission from Sprengel, A., Lill, P., Stegemann, P., Bravo-Rodriguez, K., Schoneweiss, E. C., Merdanovic, M., Gudnason, D., Aznauryan, M., Gamrad, L., Barcikowski, S., et al. (2017) Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions. Nat. Commun. 2017, 8, 14472. Copyright 2017 Springer Nature. B) Design of a DNA tetrahedron with an overhang for siRNA. C) Up to six ligands can be displayed on the tetrahedron in a spatially-controlled fashion. Reprinted (Adapted or Reprinted in part) with permission from Lee, H., Lytton-Jean, A. K. R., Chen, Y., Love, K. T., Park, A. I., Karagiannis, E. D., Sehgal, A., Querbes, W., Zurenko, C. S., Jayaraman, M., et al. (2012) Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nat. Nanotechnol. 7, 389-393. Copyright 2012 Springer Nature. D) Design of a DNA tile with a bioactive peptide attached to one of the strands. E) Self-assembly of the tile into a DNA nanotube with multivalent presentation of the RGDS signal. Reprinted (Adapted or Reprinted in part) with permission from Stephanopoulos, N., Freeman, R., North, H. A., Sur, S., Jeong, S. J., Tantakitti, F., Kessler, J. A., Stupp, S. I. (2015) Bioactive DNA-Peptide Nanotubes Enhance the Differentiation of Neural Stem Cells Into Neurons. Nano Lett. 15, 603-609. Copyright 2015 American Chemical Society. Drug delivery is another area where the three-dimensional presentation of bioactive ligands on DNA scaffolds can play a significant role. In 2012 the Anderson lab demonstrated this property by using a self-assembled DNA tetrahedron as a scaffold to present targeting peptides in order to deliver siRNA bound to the structure.23 The tetrahedron could attach up to six ligands, and several cell-penetrating peptides showed siRNA silencing activity (Figure 3B,C). Although the authors chose to use folate as a targeting ligand instead of a peptide due to its enhanced potency, their scaffold also allowed them to tune the number and 3D presentation of this signal. Gene knockdown activity peaked at three ligands, clustered on one face of the tetrahedron, demonstrating that DNA scaffolds are ideal for rapidly screening various assemblies of bioactive ligands. Interestingly, the reasons the authors ultimately opted for folate as the ultimate targeting agent was because the best performing peptides were highly cationic cell-penetrating epitopes, leading to aggregation with the negatively charged DNA structures. This drawback highlights the novel challenges at the intersection of these two molecular classes, but using anionic or neutral peptides in the future should allow for spatially-controlled targeting on DNA nanostructures. In 2015, the Stupp group demonstrated that DNA nanotubes could be used to present a dense array of bioactive peptides and guide the differentiation of neural stem cells.24 A nanotube comprised of DNA tiles was used to display the integrin-binding peptide RGDS with a spacing of 4 nm in one direction and 14 nm in the other, by co-assembling a peptide-DNA conjugates with the other structural strands of the tube (Figure 3D,E). Neural stem cells (NSCs) readily adhered to glass surfaces coated with these nanotubes and enhanced their differentiation into neurons, while suppressing the generation of undesired astrocytes. Interestingly, the nanotube morphology was critical to the bioactivity of this system; surfaces coated with unstructured DNA aggregates bearing the RGDS signal suppressed astrocyte generation (due to the peptide) but did not enhance neuronal differentiation. The DNA was critical for decoupling the nanostructured morphology from the peptide signal, by simply omitting one of the strands that make up the nanotube, while including the RGDS-DNA conjugate. With peptide nanomaterials the cell signaling domain is of comparable (or greater) size to the self-assembly motif, making it difficult to systematically decouple the peptide’s activity from the structures shape. Moreover, approaches


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like DNA origami can make structures with tunable diameters, or shapes not possible with peptide assembly alone, so combining these two materials will enable powerful structure-activity studies not currently possible. Future peptide-DNA materials can be elaborated by incorporating multiple different peptides, or controlling their spacing to further guide bioactivity. Dynamic control over ligand presentation, stiffness, and nanoscale morphology in biomaterials. The extracellular matrix (ECM) is a complex environment that provides both mechanical and chemical signals that are critical for guiding cell behavior. Recapitulating this milieu with synthetic materials has great potential for regenerative medicine and tissue engineering, and a whole host of hydrogels based on self-assembling peptides or DNA alone (among many other materials) have been developed to meet this need. The natural ECM is a highly dynamic environment, with temporal changes in ligand presentation and mechanical properties playing a central role in cell behavior.25, 26 However, recapitulating this complexity in designed biomaterials is challenging, especially if independent and reversible control of multiple properties, over multiple cycles, is necessary. Peptide-DNA biomaterials are an excellent candidate for the construction of such materials, as they can merge the programmable hybridization properties of DNA with the bioactive effects or unique structural properties of peptides. In 2017, the Stupp lab demonstrated these principles by using DNA as a functional linker to control peptide signals on a non-bioactive surface.27 Alginate-coated glass surfaces were modified with ssDNA handles, which could immobilize RGDS through the peptide-DNA (“P-DNA”) complement (Figure 4A). The RGDS-DNA strand also contained at single-stranded toehold region, so addition of a displacement strand removed the signal, regenerating the surface strand (Figure 4B). In this way, it was possible to control the adhesion and spreading of fibroblast cells on the surface over multiple cycles, without degradation of performance, and with an exceptionally mild trigger (i.e. a soluble DNA strand). The DNA could also be used as a molecular ruler to space out RGDS and a synergistic signal, PHSRN, to further enhance bioactivity, akin to the synbody and other examples described above (Figure 4C).

Figure 4: Dynamic control over ligand presentation using DNA. A) DNA can be used to tether a bioactive peptide like RGDS onto a non-bioactive surface, allowing cells to adhere and spread. B) Strand displacement can be used to reversibly add and remove the RGDS signal over multiple cycles. C) The surface strand can also serve as a “molecular ruler” to co-localize two peptide signals that work synergistically together at a specific distance. D) Attaching two different strands to the surface allows for orthogonal control of multiple signals using programmable displacement



strands. Reprinted (Adapted or Reprinted in part) with permission from Freeman, R., Stephanopoulos, N., Alvarez, Z., Lewis, J. A., Sur, S., Serrano, C. M., Boekhoven, J., Lee, S. S., Stupp, S. I. (2017) Instructing cells with programmable peptide DNA hybrids. Nat. Commun. 8, 15982. Copyright 2017 Springer Nature. The authors then demonstrated a key advantage of DNA for ECM materials: orthogonal control of multiple signals via sequence-specific strand displacement (Figure 4D). Two peptide ligands—the laminin-mimetic IKVAV and a peptide that recapitulates the activity of FGF-2—were tuned orthogonally, leading to migration and proliferation of neural stem cells cultured on the surfaces. The sequence-specific trigger strands were like control knobs that could be turned on and off independently and tunably, allowing for a smooth presentation of each signal. In principle, three or more signals (including full-length proteins) could be controlled through this method, enabling truly customizable ECM materials that can be actuated by mild and reversible triggers (soluble DNA molecules). Although beyond the scope of this review, we also highlight that peptides tethered to a surface through DNA linkers can also be used as programmable probes of the forces cells exert on their environment.28-31 DNA is an ideal linker for this purpose, with a wellunderstood relationship between the length and sequence of a hybridized duplex and the force necessary to break it; interfacing this capability with cell receptors through the use of binding peptides allows for the elucidation of cell-matrix biophysics. Future work can combine dynamic control of multiple peptide signals with multiplexed force probes to tease apart subtle cellular processes with unprecedented precision. Although peptide-based hydrogels have been used extensively in tissue engineering,3 only recently has DNA been introduced in order to reversibly control the crosslinking of these materials.32, 33 These hydrogels demonstrated DNA-dependent properties—like degradation if (1) nuclease enzymes were added, or (2) the temperature was increased beyond the melting point of the duplexes—but no biological effect was demonstrated as a result of this reversibility. In 2018, the Luijten and Stupp groups demonstrated the reversible crosslinking of self-assembled peptide hydrogels using DNA, and tied these changes to biological effects on cells from the central nervous system.34 Peptide amphiphiles (PAs)—which have extensive use as nanostructured ECM-mimetic biomaterials35-37—were synthesized bearing oligonucleotide (both DNA and PNA) handles; co-assembling these fibers at 1-10 mol% with unfunctionalized PA molecules resulted in crosslinking when mixed with fibers bearing complementary DNA sequences (Figure 5A). The authors surmised that the fibers would be randomly crosslinked (as is typically found in PA hydrogels gelled via ionic screening), and that these interconnections could be broken in a tunable fashion with displacement strands in order to tune the stiffness.


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Figure 5: Reversible control of supramolecular assembly and stiffness. A) Peptide amphiphiles (PAs) co-assembled with PA-DNA conjugates create nanofibers with a dense presentation of complementary DNA strands. B) The fibers segregate into DNA-rich bundles, and DNA-poor individual fibers, a result borne out by molecular dynamics simulations as well (C). D) Astrocytes cultured on individual fibers resulted in a naïve morphology, whereas those on bundled fibers were reactive; switching to individual fibers using a displacement strand in turn switched the morphology of the astrocytes back to naïve. E) Self-complementary, charged peptides could also be used to mimic DNA and create bundles of fibers, enabling hierarchical assembly in the absence of DNA. Reprinted (Adapted or Reprinted in part) with permission from Freeman, R., Han, M., Alvarez, Z., Lewis, J. A., Wester, J. R., Stephanopoulos, N., McClendon, M. T., Lynsky, C., Godbe, J. M., Sangji, H. et al. (2018) Reversible self-assembly of superstructured networks. Science 362 808-813. Copyright 2018 The American Association for the Advancement of Science. Contrary to these expectations, however, the PA-DNA hydrogels were comprised of thick bundles enriched in the DNA-modified molecules, with single fibers surrounding them (Figure 5B,C). This phenomenon suggested that the oligonucleotide-bearing monomers could redistribute between fibers as a result of hybridization with complementary molecules, a conclusion borne out by extensive coarse-grained molecular dynamics simulations of the system. The bundling was DNA-specific, and could be reversed over multiple cycles through strand displacement reactions or by cycling the temperature. Such control of hierarchical self-assembly is highly reminiscent of natural ECM proteins like collagen, but this work was the first demonstration of reversible control in a self-assembled peptide matrix. Critically, the bundled fiber hydrogels were roughly an order of magnitude stiffer than unbundled fibers, both in bulk and at the nanoscale. When cortical astrocytes were cultured on surfaces bearing the DNA-modified PAs, their morphology could be switched between quiescent and reactive (the latter of which impedes regeneration after damage, e.g. in spinal cord injury) by modulating the nanoscale morphology and local stiffness of the fibers (Figure 5D). This effect relied on the bundled architecture, as the change was not seen on non-bundled substrates of similar stiffness (or on



even stiffer glass substrates), demonstrating that DNA-mediated tuning of hydrogel structure was a powerful method for guiding cell behavior in regenerative medicine. Interestingly, the reversible bundling demonstrated in this work could be recapitulated in the absence of DNA using chargecomplementary peptide molecules that dimerize in an antiparallel fashion (Figure 5E), suggesting that DNA-modified materials could be used to design materials that could later be translated to entirely oligonucleotide-free analogues. Conclusions and future directions. The examples presented in this review demonstrate the extraordinary power of peptide-DNA hybrid materials for nanotechnology, biology, and medicine. The structural programmability of DNA, along with its dynamic and sequence-specific properties, is a powerful complement to the bioactivity and structural diversity of peptides. The field of peptide-DNA bionanotechnology is still in its relative infancy, however, with great potential for future materials. DNA scaffolds modified with multiple peptides might be able to recapitulate the function of full-length proteins, presenting a few functional peptides to create tightly-binding interfaces or even catalytic active sites. The DNA scaffold would effectively recapitulate the bulk of the protein sequence (which is responsible for folding in order to stabilize a few key residues responsible for bioactivity), and allow novel structures not possible with native proteins. DNA can also be used to control peptide conformations by tethering the latter to multiple points on a scaffold in order to enhance their activity (as outlined in a number of key reports not covered herein),38-41 further mimicking natural proteins. Peptide-DNA hybrids will also play an increasing role in biomaterials when independent control of a large number of signals is necessary, especially when control over stiffness, or nanoscale morphology, is also desired.

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