International Journal of Molecular Sciences

Review Supramolecular Architectures of Nucleic Acid/Peptide Hybrids

Sayuri L. Higashi 1, Normazida Rozi 2, Sharina Abu Hanifah 2 and Masato Ikeda 1,3,4,5,*

1 United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; [email protected] 2 Department of Chemical Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi 43600, Selangor, Malaysia; [email protected] (N.R.); [email protected] (S.A.H.) 3 Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 4 Center for Highly Advanced Integration of Nano and Life Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 5 Institute for Glyco-Core Research (iGCORE), Gifu University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan * Correspondence: [email protected]; Tel.: +81-58-293-2639



Received: 16 November 2020; Accepted: 9 December 2020; Published: 12 December 2020


Abstract: Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.

Keywords: supramolecular architectures; self-assembly; nanostructures; nucleic acids; peptides; molecular hybrid

1. Introduction The construction of supramolecular architectures comprising nucleic acids as well as peptides (including oligopeptides and polypeptides in this paper) has greatly progressed recently [1–3]. Particularly, deoxyribonucleic acid (DNA) nanotechnology, a concept that was introduced by Seeman in the 1980s [4,5], has greatly expanded the range of rationally designed nanostructures (Figure1A). For instance, the DNA origami technology, which was invented by Rothemund [6,7], is widely accepted as a promising strategy to construct supramolecular architectures with custom nanoscale shapes via the bottom-up approach. Therein, a long single-stranded DNA (ssDNA) was folded by the addition of hundreds of short ssDNAs, namely, staple strands, followed by one-pot thermal annealing to obtain the rationally designed shape of the supramolecular architectures (the DNA origami). Apart from the DNA origami technology, the DNA tiles and bricks (in which the long ssDNAs are not prerequisite but the sequence-designed ssDNAs with the characteristic structural motif like the holiday junction [8–11] are required) offer supramolecular architectures with various morphology such as fibers [12–14], tubes [15–20], and polyhedrons [21–23]. Evidently, the high fidelity of the predictable Watson–Crick (WC) base pairings and the regular formation of the well-defined double-stranded DNA (dsDNA) structures are indispensable to the design and construction of such beautiful supramolecular architectures based on DNA nanotechnology.

Int. J. Mol. Sci. 2020, 21, 9458; doi:10.3390/ijms21249458 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9458 2 of 25 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 25

TheseThese DNADNA nanostructures nanostructures o ffofferer a varietya variety of of bio-applications, bio-applications, such such as scaas ffscaffoldsolds for enzymesfor enzymes in the in constructionthe construction of bioreactors of bioreactors [24,25 [24,25],], vehicles vehicles for drug for delivery drug delivery [26], biosensors [26], biosen [27sors], and [27], robotics and robotics [28–31]. Despite[28–31]. theseDespite promising these promising functionsand functions advantages and ofadvantages DNA nanostructures, of DNA nanostructures, several drawbacks several are directlydrawbacks related are todirectly the intrinsic related propertiesto the intrinsic of DNA. properties One of theof DNA. most fundamentalOne of the most drawbacks fundamental is the relativelydrawbacks low is stabilitythe relatively of DNA low against stability nucleases of DNA that against exist rathernucleases abundantly that exist under rather physiological abundantly conditionsunder physiological or in biological conditions fluids [32 or,33 ].in Furthermore, biological severalfluids [32,33]. DNA nanostructures Furthermore, require several high DNA salt concentrations,nanostructures whichrequire are occasionallyhigh salt concentrations, incompatible with which biosystems are occasionally and/or deviate incompatible significantly fromwith thebiosystems standard and/or salt concentration deviate significantly for biological from fluids the standard or cell culture salt concentration media [34]. However,for biological to overcome fluids or thesecell culture drawbacks, media a [34]. variety However, of strategies, to overcome including thes moleculare drawbacks, hybrid a variety approaches of strategies, that are describedincluding herein,molecular has hybrid been proposed approaches and that investigated are described [35–40 herein,]. has been proposed and investigated [35–40]. TheThe self-assemblyself-assembly of of oligopeptides oligopeptides or or polypeptides polypeptides is also is also an actively an actively investigated investigated research research topic intopic the in field the field of biomolecular of biomolecular science science (Figure (Figure1B) [1B)41 –[41–43].43]. For For example, example, the the self-assembled self-assembled fibrousfibrous nanostructuresnanostructures ofof proteins,proteins, suchsuch asas actinactin filamentsfilaments andand microtubules,microtubules, areare keyskeys toto cellularcellular movementmovement andand elasticityelasticity [ 44[44].]. Such Such fibrous fibrous self-assembled self-assembled nanostructures nanostructures of peptidesof peptides are presentare present both both inside inside and outsideand outside living living cells [45cells]. Particularly,[45]. Particularly, amyloid amyloid fibril formations fibril formations are beneficial are beneficial research research targets because targets ofbecause their severeof their pathological severe pathological outcomes outcomes [46]. Very [4 recently,6]. Very the recently, detailed the structure detailed of structure the amyloid- of theβ peptideamyloid- inβ peptide the potentially in the potentially pathological pathological self-assembled self-assembled state was state successfully was successfully solved solved at the atomat the levelatom bylevel cryogenic by cryogenic transmission transmission electron electron microscopy microscopy (cryoTEM) (cryoTEM) techniques techniques [47]. As [47]. shown As shown there, mostthere, of most the amyloidof the amyloid fibril comprisedfibril comprised the β-sheet the β-sheet structures, structures, that is, that the is, cross- the cross-β structures.β structures. In the In cross-the cross-β structuralβ structural motif, motif, the the peptide peptide strands strands folded folded into into (at (at least least partially) partially) elongated elongatedβ β-strands-strands toto self-assembleself-assemble intointo (or(or stack)stack) fibrousfibrous structuresstructures inin whichwhich thethe longlong axisaxis ofof thethe peptidepeptide strandsstrands waswas perpendicularperpendicular toto thatthat ofof thethe fibrousfibrous structures.structures. Conversely, veryvery recently,recently, self-assembledself-assembled fibrousfibrous nanostructuresnanostructures comprisingcomprising anan αα-helix-folded-helix-folded peptidepeptide (the(the cross-cross-αα structures)structures) werewere observedobserved forfor thethe firstfirst timetime [[48,49].48,49]. ThisThis uniqueunique findingfinding andand thethe otherother structuralstructural motifsmotifs ofof thethe peptidepeptide self-assembliesself-assemblies underunder investigation,investigation, suchsuch asas thethe coiled-coilcoiled-coil structuresstructures [[50–52],50–52], couldcould furtherfurther elucidateelucidate thethe designdesign ofof supramolecularsupramolecular peptidepeptide architectures.architectures.

FigureFigure 1.1. ((AA)) DNADNAand and ( B(B)) peptide peptide nanotechnologies. nanotechnologies. RepresentativeRepresentative supramolecularsupramolecular architecturesarchitectures (nanostructures)(nanostructures) andand theirtheir typicaltypical component component structuralstructural motifs motifs are are shown. shown.

TheThe aboveabove brieflybriefly mentioned mentioned supramolecular supramolecular architectures architectures that that are are composed composed of nucleicof nucleic acids acids or peptidesor peptides could could show show several several advantages advantages in the in research the research fields offields supramolecular of supramolecular architectures. architectures. First of all,First biomolecules of all, biomolecules bear potential bear potential biocompatibility biocompatibility and biodegradability and biodegradability as well as as they well are as versatilethey are andversatile sustainable. and sustainable. In addition, In reliableaddition, automatic reliable automatic synthesis forsynthesis nucleic for acids nucleic and peptidesacids and have peptides been have been established [53–56] so that those with designated length and sequence can be synthesized

Int. J. Mol. Sci. 2020, 21, 9458 3 of 25 established [53–56] so that those with designated length and sequence can be synthesized and are even commercially available. These well-established synthetic technologies have allowed for the progress of the research fields. Despite the rapid progress in the development of supramolecular architectures that are composed solely of nucleic acids or peptides, the combination of the two biomolecules for the development of hybrid supramolecular architectures remains less explored. Therefore, this review non-exhaustively aims to highlight recently published examples to underscore hierarchical structures and the promising (future) applications of supramolecular architectures that comprised both nucleic acids and peptides by dividing them into two sections: Section2 (nucleic acid /oligopeptide hybrids) and Section3 (nucleic acid/polypeptide (protein) hybrids), based on their component peptides, oligopeptides (generally <~20 amino acids (aa) and peptides that are obtained by solid-phase peptide synthesis without ligations or gene expression (recombinant) technology) or polypeptides (proteins), by referring to “peptides with <~10–20 residues as oligopeptides and those with more residues as polypeptides” [57].

2. Nucleic Acid/Oligopeptide Hybrids

2.1. Non-Covalently Conjugated Nucleic Acid/Oligopeptide Hybrids In this subsection, we introduce supramolecular architectures that are constructed from nucleic acids and oligopeptides (without any covalent conjugation between them) in which the non-covalent bond interactions between them are in turn indispensable. On the basis of the final morphology (spherical, fibrous, etc.) of the constructed supramolecular architectures, we further discussed them in several subsubsections (Sections 2.1.1–2.1.3). In most of the examples, the oligopeptides bearing nucleic acid-binding domains derived from nucleic acid-binding proteins or simple cationic peptide domains were fused to self-assembling peptides with or without spacers to obtain the constituent oligopeptide derivatives. The inherent structures, e.g., one-dimensional and circular, of nucleic acids and/or their supramolecular nanostructures are used as templates for directing the formation of hybrid supramolecular architectures. Consequently, the sizes, shapes, and stoichiometric compositions of the final hybrid supramolecular architectures are difficult to predict. Nevertheless, rather monodispersed and uniformly fabricated supramolecular architectures have been occasionally constructed by exploiting the recent progress in DNA and peptide nanotechnology. In the following subsubsections, we highlighted some recent but not comprehensive reports [24] on such well-defined supramolecular architectures.

2.1.1. Spherical Supramolecular Architectures A series of well-organized spherical supramolecular architectures of nucleic acids and oligopeptides were recently developed by Ni and Chau [58–60]. For instance, they developed a parapoxvirus mimetic ellipsoid, that is, “nanococoons” (approximately 65 45 nm2, as estimated by atomic force microscopy × (AFM) and TEM), by the synergistic co-assembly of a self-assembling peptide derivative (K3C6SPD: KKK-C6-WLVFFAQQ-GSPD, 15 aa, and a C6 spacer; Figure2A) and a plasmid DNA (pDNA, 4.7 kbp) [ 58]. The K3C6SPD peptide contains a DNA-binding cationic tripeptide domain (K3), a self-assembled β-sheet domain (a hydrophobic segment that was derived from an amyloid-β peptide {Aβ (17–21)}), and a hydrophilic segment at the C-terminus, which was designed according to viral capsid proteins, for controlled water dispersibility. As illustrated in Figure2B, K3C6SPD can independently self-assemble into antiparallel β-sheet bilayer-based filamentous nanoribbons (approximately 4 12 nm2, as estimated × by AFM and TEM). Upon adding pDNA in an appropriate n/p ratio of 20, K3C6SPD was simultaneously coated around pDNA and eventually formed the nanococoons (Figure2C(a)). The unique structural model of the nanococoons was proposed, as shown in Figure2C(b), based on the TEM images and the other analyses. Noteworthily, the nanococoons exhibited significant tolerance against degradations by enzymes, e.g., trypsin or chymotrypsin as the proteases and deoxyribonuclease I (DNase I) as the nucleases, most probably because of the strong association between the self-assembled K3C6SPD and pDNA and the compactly assembled total structure. Int. J. Mol. Sci. 2020, 21, 9458 4 of 25 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 25

FigureFigure 2.2. (A(A)) ChemicalChemical structure structure of of the the self-assembling self-assembling peptide peptide derivatives derivatives containing containingK3, K a3 self-, a assemblingself-assemblingβ-sheet β-sheet domain, domain, and and a hydrophilica hydrophilic segment segment at at the the C-terminusC-terminus forfor controlled water water dispersibility.dispersibility. (B) SchematicSchematic illustration of the self-assembl self-assemblyy of of the peptide derivatives derivatives to to form form the the nanoribbons.nanoribbons. (C(C)() a()a Schematic) Schematic illustration illustration of theof co-assemblythe co-assembly of the of peptide the peptide derivatives derivatives with plasmid with DNAplasmid (pDNA) DNA to(pDNA) form a varietyto form of a hybridvariety nanostructures. of hybrid nanostructures. (b) Cross-sectional (b) Cross-sectional view of the nanococoons.view of the Adaptednanococoons. from Adapted [58]. Copyright from [58]. 2014 Copyright American 2014 Chemical American Society. Chemical Adapted Society. from [Adapted59]. Copyright from [59]. 2017 JohnCopyright Wiley 2017 and SonsJohn Publisher.Wiley and Sons Publisher.

Thereafter,Thereafter, thethe samesame researchresearch groupgroup successfully tuned the the morphology, morphology, stability, stability, and and cellular cellular uptakeuptake (gene(gene transfection) transfection) effi ciencyefficiency of the of peptide–DNA the peptide–DNA virus-mimicking virus-mimicking complexes complexes by modulating by themodulating amino acid the sequence amino acid in thesequence central in self-assembling the central self-assemblingβ-sheet segment β-sheet (originally, segment 8 aa:(originally, WLVFFAQQ) 8 aa: ofWLVFFAQQ) the peptide componentsof the peptide [59 ].components Exhaustively, [59]. six Exha differentustively, central six segmentsdifferent thatcentral comprised segments leucine that (L)comprised and/or alanineleucine (A)(L) and/or (L8: LLLLLLLL, alanine (A)L6 (L8: LLLLLL,: LLLLLLLL,L4: LLLL,L6: LLLLLL,A8: AAAAAAAA, L4: LLLL, A8: A6:AAAAAAAA, AAAAAA, andA6: (AAAAAA,L2A2)2: LLAALLAA, and (L2A2)2 from: LLAALLAA, 8 to 4 aa) from were 8 investigated to 4 aa) were (Figure investigated2A), and (Figure it was 2A), observed and it thatwas increasingobserved that the hydrophobicityincreasing the hydrophobicity of the amino acid of sidethe chainsamino (fromacid side A to chains L) and (from the length A to (fromL) and 4 tothe 8 aa)length of the (from central 4 segmentto 8 aa) ofof the the peptides central enhanced segmentthe of inter-nanofibrilthe peptides enhanced association the and inter-nanofibril thus controlled theassociation final morphology and thus of controlled the peptide the/DNA final virus-mimicking morphology of complexes. the peptide/DNA Various morphologies, virus-mimicking e.g., thecomplexes. tangles for VariousA6/DNA, morphologies, the nanofibril e.g., networks the tangles for A8/ DNA,for A6 the/DNA, striped the nanococoons nanofibril fornetworksL4 or L6 foror (A8L2A2/DNA,)2/DNA, the striped and the nanococoons coexistence of for nanofibrils L4 or L6 or and (L2A2 nanococoons)2/DNA, and for L8 the/DNA, coexistence were observed of nanofibrils in the nand/p ratio nanococoons of 20 (Figure for L82C(a))./DNA, Expectedly, were observed the strength in the n/p of theratio inter-nanofibril of 20 (Figure 2C(a)). association Expectedly, regulated the thestrength stability of ofthe the inter-nanofibril structure and DNAassociation protection, regulated as well the as stability the gene of transfection the structure efficiency and ofDNA the nanococoons.protection, as Fortunately,well as the gene such transfection a strong inter-nanofibril efficiency of the association nanococoons. that increased Fortunately, the stabilitysuch a strong of the nanococooninter-nanofibril structures association did not that exert increased significantly the stab negativeility of impact the nanococoon on the gene structures transfection did effi notciency exert in theirsignificantly study, and negative the strategy impact might on the be beneficialgene transfection for exploring efficiency gene deliveryin their systems.study, and the strategy mightVery be beneficial recently, thefor exploring same research gene delivery group constructed systems. pH-responsive peptide/DNA complexes, that is,Very “nanoberries,” recently, the with same a structurallyresearch group lower constructed aspect ratio pH-responsive (a more planer peptide/DNA structure) [60 complexes,] than that ofthat the is, nanococoons “nanoberries,” described with a structurally above. These lower nanoberries aspect ratio comprised (a more planer a peptide structure) (H4K [60]5HC thanBzlC BzlthatH : HHHHKKKKK-C12-LLHCof the nanococoons describedBzlC Bzlabove.HLLGSPD; These 21nanoberries aa, including comprised an unnatural a peptide amino ( acid,H4K5 “benzylatedHCBzlCBzlH: cysteineHHHHKKKKK-C12-LLHC (CBzl),” and a C12BzlC spacer;BzlHLLGSPD; Figure 213A), aa, and including pDNA an (4.7 unnatural kbp), asamino shown acid, in “benzylated Figure3B. ThecysteineH4K 5(CHCBzlBzl),”C andBzlH apeptide C12 spacer; was rationallyFigure 3A), designed and pDNA from similar(4.7 kbp), peptides as shown and optimizedin Figure to3B. install The pHH4K responsiveness5HCBzlCBzlH peptide through was the rationally introductions designed of histidine from simi residueslar peptides at the DNA-binding and optimized domain, to install as wellpH asresponsiveness the self-assembling through central the domainintroductions (to promote of histid endosomaline residues escape at the inside DNA-binding living cells) domain, and the aromatic as well residuesas the self-assembling of CBzl for the central formation domain of the (to self-assembled promote endosomal nanostructure. escape inside Indeed, livingH4 Kcells)5HC BzlandCBzl theH 2 wasaromatic assembled residues into uniformof CBzl for nanodiscs the formation (approximately of the 7 self-assembled7 nm (width nanostructure.length), as estimated Indeed, × × byH4K TEM,5HCBzl andCBzl approximatelyH was assembled 4 nm into (height), uniformas nanodiscs estimated (approximately by AFM) comprising 7 × 7 nm2β (width-sheet structures.× length), as estimated by TEM, and approximately 4 nm (height), as estimated by AFM) comprising β-sheet

Int. J. Mol. Sci. 2020, 21, 9458 5 of 25

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 25 Upon complexation with pDNA, the peptide/DNA complexes, which exhibited the morphology of thestructures. nanoberries Upon (approximatelycomplexation with 50 pDNA, nm, as th estimatede peptide/DNA by TEM), complexes, were obtained which exhibited (Figure3 C,D);the morphology of the nanoberries (approximately 50 nm, as estimated by TEM), were obtained (Figure a virus-mimetic structural model of the nanoberries, in which the core DNA was wrapped by nanodiscs, 3C,D); a virus-mimetic structural model of the nanoberries, in which the core DNA was wrapped was proposed. Crucially, the nanoberries exhibited a distinct structural transition from the compactly by nanodiscs, was proposed. Crucially, the nanoberries exhibited a distinct structural transition packed nanoberries to loosen complexes with heterogeneous sizes due to the acidic pH shift (from 7 from the compactly packed nanoberries to loosen complexes with heterogeneous sizes due to the to 5). Subsequently, the encapsulated DNA was released via polyanion exchange with heparin acidic pH shift (from 7 to 5). Subsequently, the encapsulated DNA was released via polyanion (anexchange anionic polysaccharide).with heparin (an Suchanionic an acidicpolysaccharide). pH shift-initiated Such an stepwiseacidic pH structural shift-initiated changes stepwise and the concurrentstructural release changes of the and encapsulated the concurrent DNA release for the of virus-mimicking the encapsulated artificial DNA for peptide the virus-mimicking/DNA nanoberries couldartificial greatly peptide/DNA contribute tonanoberries gene delivery could like greatly natural contribute viruses. to Indeed, gene delivery the transfection like natural ability viruses. ofthe nanoberriesIndeed, the was transfection demonstrated ability by of an the enhanced nanoberries expression was demonstrated of the green by fluorescent an enhanced protein expression (GFP) of that wasthe encoded green fluorescent into the encapsulated protein (GFP) pDNA. that was encoded into the encapsulated pDNA.

FigureFigure 3. 3.( A(A)) ChemicalChemical structure of of the the self-assembling self-assembling peptide peptide derivative, derivative, H4K5HHC4KBzl5HCCBzlBzlH. C(BBzl) H. Schematic illustration of a nanodisc that was constructed via the self-assembly of H4K5HCBzlCBzlH. (B) Schematic illustration of a nanodisc that was constructed via the self-assembly of H4K5HCBzlCBzlH. (C)( SchematicC) Schematic illustration illustration and and ( D(D)) representative representative TEM image image (magnifi (magnifieded in in inset) inset) of ofthe the co-assembly co-assembly between H4K5HCBzlCBzlH with pDNA to form the nanoberries. Adapted from [60]. Copyright 2020 between H4K5HCBzlCBzlH with pDNA to form the nanoberries. Adapted from [60]. Copyright 2020 JohnJohn Wiley Wiley and and Sons Sons Publisher. Publisher.

2.1.2.2.1.2. Fibrous Fibrous Supramolecular Supramolecular Architectures Architectures SinceSince the the early early stage stage of of supramolecular supramolecular chemistrychemistry and and self-assembly self-assembly [61,62], [61,62 ],fibrous fibrous tobacco tobacco mosaicmosaic virus virus (TMV) (TMV) has has been been one one of of the the mostmost attractiveattractive and referenced referenced fibrous fibrous bio-supramolecular bio-supramolecular architecturesarchitectures comprising comprising a single-strandeda single-stranded genetic genetic ribonucleic ribonucleic acid acid (ssRNA) (ssRNA)(approximately (approximately 64006400 nt) thatnt) is surroundedthat is surrounded by constituent by constituent coat proteins coat protei [63]. Veryns [63]. recently, Very therecently, bio-supramolecular the bio-supramolecular architecture of TMVarchitecture was applied of TMV to was construct applied morphologically to construct morp uniquehologically supramolecular unique supramolecular architectures architectures by combining it withby combining the DNA origami it with nanotechnology. the DNA origami For nanotechnology. example, Wang andFor coworkersexample, recentlyWang and designed coworkers a DNA

Int. J. Mol. Sci. 2020, 21, 9458 6 of 25 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 25 origamirecently nanotube designed that a DNA was tethered origami tonanotube an ssRNA that strand was tethered via hybridization to an ssRNA with strand a complementary via hybridization DNA strandwith that a complementary was connected DNA to the strand nanotube that [was64]. connected The in situ to assembly the nanotube of the [64]. TMV The coat in proteinssitu assembly formed semi-artificialof the TMV TMVcoat proteins nanostructures formed semi-artificial at designated TMV positions nanostructures of the DNA at origamidesignated nanotubes, positions as of shown the in FigureDNA origami4A. More nanotubes, recently, as they shown successfully in Figure controlled 4A. More therecently, spatiotemporally they successfully precise controlled assembly the of thespatiotemporally TMV protein more precise on theassembly surface of of the a DNA TMV origamiprotein trianglemore on inthe a similarsurface designof a DNA [65], origami although theytriangle applied in a toeholdsimilar design mediated [65], strandalthough displacement they applied (TMSD) a toehold reaction, mediated as outlinedstrand displacement in Figure4 B. (TMSD) reaction, as outlined in Figure 4B. In detail, the short DNA strands that protruded from the In detail, the short DNA strands that protruded from the DNA origami triangle were designed to DNA origami triangle were designed to bind a specific sequence of the ssRNA strand, which was bind a specific sequence of the ssRNA strand, which was captured at programmed points on the captured at programmed points on the surface of the triangle. The programmed points functioned surface of the triangle. The programmed points functioned as “locks” to stop the assembly of the TMV as “locks” to stop the assembly of the TMV protein. Thereafter, a complementary short DNA strand protein. Thereafter, a complementary short DNA strand containing a toehold region released ssRNA containing a toehold region released ssRNA via the TMSD reaction, thereby resulting in the TMV viaprotein the TMSD assembly reaction, up to thereby the following resulting “locks” in the point. TMV This protein strategy assembly allowed up the to theprecise following control “locks”of a point.specific This protein strategy assembly allowed within the precise the kinetic control system of a specific containing protein a travel assembly pathway, within the the positions, kineticsystem and containingthe assembly a travel stoichiometries, pathway, the positions,which was and crucial the assemblyto the construction stoichiometries, of further which sophisticated was crucial and to the constructionfunctional ofhybrid further nanostructures. sophisticated and functional hybrid nanostructures.

FigureFigure 4. (4.A )((Ai) Schematic(i) Schematic illustration illustration of theof formationthe formation of a of semi-artificial a semi-artificial tobacco tobacco mosaic mosaic virus virus (TMV) nanostructure(TMV) nanostructure at a designated at a designated position posi oftion the DNAof the origamiDNA origami nanotube nanotube and (andii) representative(ii) representative TEM imagesTEM ofimages the in of situ the assembled in situ assembled protein nanotubes protein nanotubes on DNA origamion DNA nanotubes origami nanotubes with one dockingwith one site. (B)(dockingi) Schematic site. (B illustration) (i) Schematic of the illustration spatiotemporally of the spatiotemporally controlled TMV controlled protein assembly TMV protein on the assembly surface of a DNA origami triangle, (ii,iii) representative TEM images of each state illustrated in (i). Scale bar: 50 nm. Adapted from [64,65]. Copyright 2018 and 2020 American Chemical Society. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 25

on the surface of a DNA origami triangle, (ii, iii) representative TEM images of each state illustrated Int. J. Mol.in (i). Sci. Scale2020 bar:, 21, 50 9458 nm. Adapted from [64,65]. Copyright 2018 and 2020 American Chemical Society. 7 of 25

To artificially create a virus-like filamentous supramolecular architecture with a well-defined length,To Stupp artificially and createcoworkers a virus-like described filamentous a distinct supramolecularstrategy, which applies architecture the self-assembly with a well-defined of the length,designed Stupp artificial and peptide-based coworkers described nanostructures a distinct (Figure strategy, 5) on which a linear applies or circular the self-assembly dsDNA, as ofa thetemplate designed [66]. artificial This sophisticated peptide-based supramolec nanostructuresular design (Figure would5) on abe linear a reminiscence or circular dsDNA,of the asaforementioned a template [ 66TMVs]. This of sophisticatedbiosystems. The supramolecular mushroom-shaped design nanostructures would be a reminiscence were constructed of the aforementionedthrough the self-assembly TMVs of biosystems. of triblock The peptide-based mushroom-shaped molecules nanostructures (PEGylated were coiled-coil constructed peptides: through theSP-CC-PEG self-assembly) containing of triblock a cationic peptide-based spermine molecules unit at one (PEGylated terminus coiled-coil (a binding peptides: site for SP-CC-PEGdsDNA); a ) containingcoiled-coil a cationicpeptide spermine(REGVAKALRAVANAL unit at one terminusHYNASALEEVADALQKVKM: (a binding site for dsDNA); a coiled-coil34 aa), peptidewhich (REGVAKALRAVANALHYNASALEEVADALQKVKM:aggregates into heptameric structures; and a long poly(ethylene 34 aa), glycol) which (PEG) aggregates chain, into which heptameric imparts structures;it with water and solubility, a long poly(ethylene as well as steric glycol) repulsion (PEG) between chain, whichthe PEG imparts clusters it in with the waterself-assembled solubility, asstructure. well as Interestingly, steric repulsion the between longer PEG the PEGwas clusterscritical to in both the self-assembled the formation structure.of the monodispersed Interestingly, thefilamentous longer PEG complexes was critical and to the both suppression the formation of am of theorphous monodispersed (not-well-ordered filamentous structure) complexes formation. and the suppressionUpon complexing of amorphous SP-CC-PEG (not-well-ordered5000 with dsDNAs structure) (150, formation. 300, 600, Upon and complexing 1200 bp)SP-CC-PEG or extended5000 withsupercoiled dsDNAs pDNAs (150, 300, (2.7, 600, 4.4, and and 1200 10.2 bp) kbp), or extendedhomogenous supercoiled supramolecular pDNAs (2.7,rod-like 4.4, andobjects 10.2 with kbp), homogenouscontrolled lengths supramolecular were successfully rod-like constructed. objects with The controlled development lengths of were this successfullykind of supramolecular constructed. Thearchitectures development in which of this the kind nucleic of supramolecular acids are enwra architecturespped in the in peptides which the and/or nucleic their acids supramolecular are enwrapped innanostructures the peptides have and /beenor their actively supramolecular investigated nanostructuresfor applications have in non-viral been actively gene delivery investigated systems for applications[67]. in non-viral gene delivery systems [67].

Figure 5.5.( A(A) Design) Design of SP-CC-PEGof SP-CC-PEG to form to mushroom-shapedform mushroom-shaped nanostructures. nanostructures. (B) Schematic (B) Schematic illustration ofillustration the formation of the of formation supramolecular of supramolecular rod-like nanostructures rod-like nanostructures through the complexation through the ofcomplexation SP-CC-PEG5000 of withSP-CC-PEG dsDNAs.5000 Adaptedwith dsDNAs. from [Adapted66]. Copyright from [66]. 2013 Co Americanpyright 2013 Chemical American Society. Chemical Society.

Ke and coworkers have demonstrated that that the the co-assembly co-assembly of of two-layered two-layered DNA DNA origami origami nanosheets (DNA(DNA TL TL nanosheets) nanosheets) and and collagen-mimetic collagen-mimetic peptides peptides obtained obtained one-dimensional one-dimensional peptide / DNApeptide/DNA hybrid nanowires hybrid nanowires (Figure6 )[(Figure68]. Specifically, 6) [68]. Specifically, three collagen-like three collagen-like triple helix-forming triple helix-forming peptides CP+ CP++ sCP++ (peptides: (PRG) (CP+7(P-Hyp-G): (PRG)7(P-Hyp-G)4(E-Hyp-G)4(E-Hyp-G)4 {45 aa},4 {45 aa},: (PRG) CP++3: (P-Hyp-G)(PRG)3(P-Hyp-G)7(PRG)73(PRG){39 aa},3 {39 and aa}, and : (PRG)sCP++3: (P-Hyp-G)(PRG)3(P-Hyp-G)4(PRG)34(PRG){30 aa};3 {30 Figure aa};6 A)Figure were investigated6A) were investigated for the construction for the construction of well-defined of hybridwell-defined nanostructures hybrid nanostructures with the DNA with TL the nanosheets. DNA TL nanosheets. It would It be would reasonable be reasonable to expect to thatexpect the 3 polyanionic DNA TL nanosheets (approximately 50 50 5 nm , estimated3 by TEM) would form that the polyanionic DNA TL nanosheets (approximately× 50× × 50 × 5 nm , estimated by TEM) would complexesform complexes with thewith cationic the cationic collagen-like collagen-like triple helicestriple bearinghelices bearing arginine-rich arginine-rich overhangs overhangs through electrostaticthrough electrostatic attractions. attractions. They initially They initially confirmed confirmed thatthe that arrangement the arrangement of CP of+ CP+on theon the DNA DNA TL nanosheetTL nanosheet was was not not structurally structurally well-ordered. well-ordered. In In sharp sharp contrast, contrast,CP CP++++ and and sCPsCP++++ bearingbearing cationic cationic (PRG) triads in both the N- and C-terminal ends co-assembled with the DNA TL nanosheets to form striped well-ordered nanowire structures. Crucially, the distances between the TL nanosheets Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 25

Int. J. Mol. Sci. 2020, 21, 9458 8 of 25 (PRG) triads in both the N- and C-terminal ends co-assembled with the DNA TL nanosheets to form striped well-ordered nanowire structures. Crucially, the distances between the TL nanosheets (the inter-sheet(the inter-sheet distances) distances) were were successfully successfully estimated estimated by by TEM TEM and and synchrotron synchrotron small- small- and and wide-angle wide-angle X-ray scattering (SAXS/WAXS) (SAXS/WAXS) and were well consis consistenttent with the length of of the the cationic cationic peptides. peptides. This indicated that the peptide helices could alig alignn perpendicularly to to the surface of the DNA TL nanosheets, as depicted in Figure 6 6B,C.B,C.

Figure 6.6. (A(A)) Sequences Sequences of theof the three three collagen-like collagen-lik triplee triple helix-forming helix-forming peptides peptides (CP+, CP (CP+++,, and CP++sCP, ++and). sCP++(B) Schematic). (B) Schematic illustration illustration of the co-assembly of the co-assembly of the two-layered of the two-layered DNA origami DNA nanosheets origami nanosheets (DNA TL (DNAnanosheets) TL nanosheets) and the collagen-mimetic and the collagen-mimetic peptides (CP++ peptidesand sCP (CP++++) to and obtain sCP++ the) one-dimensional to obtain the one-dimensionalpeptide/DNA hybrid peptide/DNA nanowires. (hybridC) Representative nanowires. TEM (C) imagesRepresentative of assembled TEM with images DNA TLof nanosheetsassembled withand CP DNA++ .TL Scale nanosheets bar: 50 nm. and Adapted CP++. Scale from bar: [68 ].50 Copyright nm. Adapted 2017 from American [68]. Copyright Chemical Society.2017 American Chemical Society. The two-dimensional, as well as the one-dimensional DNA origami nanostructures, were organized by exploitingThe two-dimensional, specific peptide as well self-assemblies. as the one-di Formensional instance, DNA Turberfield origami nanostructures, and coworkers were have organizedvery recently by exploiting reported thatspecific two peptide distinct self-assemblies. one-dimensional For DNA instance, origami Turberfield nanostructures and coworkers can be haveconnected very recently by the specific reported formation that two of distinct peptide one-dimensional coiled-coil heterodimers DNA origami (CC-Di-EK nanostructures: Ac-GEIAALEQ- can be connectedENAALEQ-KIAALKW-KNAALKQG: by the specific formation 30 aa,of CC-Di-KEpeptide :coiled-coil Ac-GKIAALKQ-KNAALKY-EIAALEQ- heterodimers (CC-Di-EK: Ac-GEIAALEQ-ENAALEQ-KIAALKW-KNAALKQG:ENAALEQG: 30 aa), as depicted in Figure7A. The overall charges30 of each peptideaa, were designedCC-Di-KE to be: Ac-GKIAALKQ-KNAALKY-EIAALEQ-ENAALEQG:close to or exactly zero at a neutral pH to avoid nonspecific 30 aa electrostatic), as depicted interactions. in Figure 7A. To optimizeThe overall the chargesformation of ofeach the connectedpeptide were heterodimer designed structure to be clos as depictede to or exactly in Figure zero7B, theat a number neutral of pH peptides to avoid ( n) nonspecificto be attached electrostatic to one side ofinteractions. each one-dimensional To optimize DNA the origami formation nanostructure of the connected was varied heterodimer from 1 to 3. structureThe TEM observationsas depicted in clearly Figure revealed 7B, the the number formation of peptides of the heterodimer, (n) to be attached and the to formation one side e ffiofciency each one-dimensionalattained approximately DNA origami 40% at nanostructuren = 3, although was it was vari

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Int.nanostructures J. Mol. Sci. 2020 , 21(micrometer-long, 9458 one-dimensional arrays) were constructed by a similar9 of 25 architectural design to that of Stephanopoulos and coworkers. They designed similar peptide coiled-coil heterodimers (EI: Ac-EIAALEK-EIAALEK-ENAALEW-EIAALEK: 28 aa, KI: Ac-KIAALKE-KIAALKE-KNAALKW-KIAALKE: 28 aa (Figure7D)), although they attached the Ac-KIAALKE-KIAALKE-KNAALKW-KIAALKE: 28 aa (Figure 7D)), although they attached the two two distinct peptides to both sides of the DNA origami cuboid nanostructure (16 19.5 32 nm3) distinct peptides to both sides of the DNA origami cuboid nanostructure (16 × 19.5 × ×32 nm3)× through through DNA–DNA hybridizations, which allowed for the formation of an elongated one-dimensional DNA–DNA hybridizations, which allowed for the formation of an elongated one-dimensional array, array, as shown in Figure7E. They also investigated the optimal number of peptides ( m) required for as shown in Figure 7E. They also investigated the optimal number of peptides (m) required for the the design and observed via AFM that the formation of long arrays comprising 30–45 cuboids occurred design and observed via AFM that the formation of long arrays comprising 30–45 cuboids occurred at m = 8 (Figure7F). Such long arrays were not observed at m = 10 and 12, and shorter oligomers at m = 8 (Figure 7F). Such long arrays were not observed at m = 10 and 12, and shorter oligomers m m (mainly(mainly< ˂1515 cuboids)cuboids) werewere mainlymainly obtainedobtained atat m << 8 (0, 1, 2, 4, and 6). This This clearly clearly indicated indicated that that m == 88 wouldwould be be optimal optimal to to obtain obtain long long one-dimensional one-dimensional arrays arrays of of the the DNA DNA origami origami cuboid cuboid nanostructure nanostructure for reasonsfor reasons that that remain remain currently currently unclear unclear according according to their to their study study [70 ].[70].

FigureFigure 7.7. ((A)) SequencesSequences of the coiled-coil heterodimers heterodimers that that formed formed the the peptides peptides ( (CC-Di-EKCC-Di-EK andand CC-Di-KECC-Di-KE)) thatthat were were conjugated conjugated to to the the oligonucleotides oligonucleotides (α, (αβ)., β(B).) Schematic (B) Schematic illustration illustration and (C and) a (representativeC) a representative TEM TEMimage image of the of theconnection connection of oftwo two distinct distinct one-dimensional one-dimensional DNA DNA origami origami nanostructuresnanostructures ( Origamis AA andand BB) )by by the the specific specific formation formation of peptide of peptide coiled-coil coiled-coil heterodimers. heterodimers. (D) (SequencesD) Sequences of the of the coiled-coil coiled-coil heterodimers heterodimers that that formed formed the the peptides peptides (EI (EI andand KIKI) that) that were were conjugated conjugated toto the the oligonucleotides oligonucleotides (DNA (DNAA Aand andB B).). ( E(E)) Schematic Schematic illustration illustration and and ( F(F)) representative representative AFM AFM images images to obtainto obtain long long one-dimensional one-dimensional arrays arrays of DNA of DNA origami origami cuboid cuboid nanostructures. nanostructures. Scale Scale bar: bar: 50 nm50 nm for Cforand C 1andµm 1 (250 μm nm(250 (inset)) nm (inset)) for F. Adaptedfor F. Adapted from [ 69from,70 ].[69,70]. Copyright Copyrigh 2019 andt 2019 2020 and American 2020 American Chemical Chemical Society. Society.

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Int. J. Mol. Sci. 2020, 21, 9458 10 of 25 2.1.3. Orthogonal Supramolecular Architectures Without Random Co-assembly Orthogonal supramolecular architectures through self-sorting phenomena to form plural 2.1.3. Orthogonal Supramolecular Architectures Without Random Co-Assembly distinct supramolecular nanostructures of molecular constituents without a random co-assembly haveOrthogonal attracted increased supramolecular attention architectures [71–74]. In throughthis context, self-sorting Seeman phenomena and coworkers to form developed plural distinct DNA supramolecularorigami nanotubes nanostructures in which the of molecularamyloid fibrils constituents were sheathed, without a and random the resultant co-assembly fibril-filled have attracted DNA increasedorigami nanotubes attention [were71–74 further]. In this organized context, Seeman into the and other coworkers DNA origami developed platforms DNA origami(two-dimensional nanotubes inplanar which monolayer the amyloid structures fibrils were that sheathed, comprised and thefour resultant flat DNA fibril-filled origami DNA hollow origami square nanotubes motifs) were via furtherDNA–DNA organized hybridizations, into the other as DNAdepicted origami in Figure platforms 8A (two-dimensional[75]. To construct planar the self-sorted, monolayer fibril-filled structures thatDNA comprised origami nanotubes, four flat DNA a staple origami DNA hollow strand square (31 motifs)nt) was via covalently DNA–DNA conjugated hybridizations, to a fibril-forming as depicted inpeptide Figure (AcTTR1-GGK8A[ 75]. To construct {TTR105–115 the self-sorted, with a tripeptide fibril-filled (GGK) DNA segment origami nanotubes,extension and a staple an acetyl DNA strandcap at (31the nt)N-terminus}; was covalently Ac-YTIAALLSPYSGGK: conjugated to a fibril-forming 14 aa) that peptide was (AcTTR1-GGK derived from {TTRthe amyloidogenic105–115 with a tripeptide protein (GGK)transthyretin segment (TTR). extension The staple and an DNA acetyl that cap conjugat at the N-terminus};ed with AcTTR1-GGK Ac-YTIAALLSPYSGGK: afforded a nucleation 14 aa) thatsite, waswhich derived was frompositioned the amyloidogenic inside the DNA protein origami transthyretin nanotube. (TTR). Notably, The staple the DNA negatively that conjugated charged withfibril-forming AcTTR1-GGK sequence afforded (AcTTR1-GGE; a nucleation site,Ac-YTIAALLSPYSGGE: which was positioned 14 inside aa) was the DNAdesigned origami to avoid nanotube. the Notably,nonspecific the interactions negatively charged between fibril-forming the DNA nanotubes sequence and (AcTTR1-GGE; the peptide Ac-YTIAALLSPYSGGE:fibrils. The fibril formation 14 aa) of wasthe AcTTR1-GGE designed to avoid peptide the nonspecificwithout DNA, interactions which indicat betweened the DNApresence nanotubes of a typical and thecross- peptideβ structure, fibrils. Thewas fibrilfirst formationrevealed by of theTEM AcTTR1-GGE and AFM observations, peptide without as well DNA, as whichX-ray indicatedfiber diffraction. the presence After ofthe a typicalthermal cross- annealingβ structure, of an M13 was firstscaffold revealed DNA by strand TEM with and AFMstaple observations, strands including as well one, as X-raywhich fiberwas diconjugatedffraction. Afterto AcTTR1-GGK, the thermal annealing the formation of an M13 scaof ffanold DNAunbranched strand withamyloid staple strandsfibril proceeded including one,simultaneously which was conjugatedthrough the to AcTTR1-GGK,addition of the the Ac formationTTR1-GGE of an peptides. unbranched The amyloidformation fibril of proceeded the fiber simultaneouslyinside the DNA through origami the nanotubes addition ofwas the indirectly AcTTR1-GGE demonstrated peptides. Theby formationseveral constructs, of the fiber e.g., inside the thedecoration DNA origami of gold nanotubes nanoparticles was indirectlyon the outer demonstrated surface of bythe several DNA constructs,origami nanotubes. e.g., the decoration Finally, a ofprogrammable gold nanoparticles DNA ondesign the outer could surface enable of the DNAprecise origami positioning nanotubes. of fibril-filled Finally, a programmableDNA origami DNAnanotubes design on could prepared enable thetwo-dimensional precise positioning planar of fibril-filledDNA origami DNA platforms, origami nanotubes which was on prepared further two-dimensionaldemonstrated by planarAFM (Figure DNA origami 8B). platforms, which was further demonstrated by AFM (Figure8B).

Figure 8.8. ((A)A) Schematic illustration and (B) a representative AFM image of amyloid fibril-filledfibril-filled DNA origami nanotubes that werewere organizedorganized ontoonto two-dimensionaltwo-dimensional DNADNA origamiorigami platforms.platforms. Scale bars: 250250 nm.nm. The colour scale bar indicates height. Ad Adaptedapted from [[75].75]. Copyright 2014 Springer Nature.

Very recently,recently, we we revealed revealed the orthogonalthe orthogonal coexistence coexistence of DNA of microspheres,DNA microspheres, which can which be obtained can be fromobtained three from ssDNAs three (30 ssDNAs nt), and (30 supramolecular nt), and supramolecular nanostructures nanostructures (helical nanofibers, (helical straight nanofibers, nanoribbons, straight andnanoribbons, flowerlike and microaggregates) flowerlike microaggregates) of semi-artificial glycopeptidesof semi-artificial comprising glycopeptides a diphenylalanine comprising (FF) a dipeptidediphenylalanine (vide infra), (FF) asdipeptide shown in (vide Figure infra),9[ 76]. as One shown of the mainin Figure factors 9 responsible[76]. One of for the such main orthogonal factors assemblyresponsible (self-sorting) for such orthogonal that was assembly induced (self-sort only bying) simple that one-pot was induced and one-step only by simple thermal one-pot annealing, and wouldone-step be thermal the distinct annealing, driving would forces ofbe thethe self-assembly,distinct driving i.e., forces the specific of the self-assembly, WC base pairings i.e., ofthe the specific DNA strandsWC base and pairings the cross ofβ -sheetthe DNA assembly strands of theand glycopeptides. the cross β-sheet Additionally, assembly the of selective the glycopeptides. degradation propensityAdditionally, (in the response selective to biostimuli, degradation such propensity as a protease, (in response DNase, orto ssDNA) biostimuli, of each such supramolecular as a protease, nanostructureDNase, or ssDNA) was retained of each and su couldpramolecular enable the constructionnanostructure of activewas retained soft nanomaterials and could with enable excellent the biofunctions.construction Weof believeactive thatsoft thenanomaterials combination ofwith self-assembling excellent biofunctions. nucleic acids andWe peptidesbelieve wouldthat the be acombination promising strategyof self-assembling for constructing nucleic such acids orthogonal and peptides supramolecular would be architectures, a promising as describedstrategy for in

Int.Int. J.J. Mol. Sci. 2020,, 21,, x 9458 FOR PEER REVIEW 1111 of of 25 25

constructing such orthogonal supramolecular architectures, as described in this review, thus thisenabling review, the thus construction enabling of the more construction complex and of more functional complex hybrid and functionalsupramolecular hybrid architectures supramolecular that architecturesare otherwise that unachievable are otherwise and unachievableyet inaccessible and [77]. yet inaccessible [77].

FigureFigure 9.9. ((A)) SchematicSchematic illustration of the orthogonal self-assemblyself-assembly of of semi-artificial semi-artificial glycopeptides glycopeptides (right(right panel)panel) and and ssDNAs ssDNAs (left panel)(left panel) to produce to hybridproduce soft hybrid materials soft that materials comprised that supramolecular comprised nanostructuressupramolecular ofnanostructures glycopeptide of and glycopeptide DNA microspheres. and DNA (microspheres.B) Representative (B) Representative confocal laser scanningconfocal microscopylaser scanning (CLSM) microscopy images (CLSM) showing images the showin orthogonalg the orthogonal coexistence coexiste of DNAnce microsphereof DNA microsphere and the supramolecularand the supramolecular nanostructures nanostructures of (a) Z-FF-Mal of (a) Z-FF-Mal,(b) Z-FF-Cel, (b) Z-FF-Cel, and (, cand) Z-FF-Lac (c) Z-FF-Lac. Scale. Scale bar: bar: 5 µ m5 forμma forand a and 10 µ m10 forμmb for,c. b Adapted,c. Adapted from from [76]. [76]. Copyright Copyright 2019 2019 John John Wiley Wiley and and Sons Sons Publisher. Publisher.

2.2.2.2. Covalently Conjugated Nucleic Acid-Oligopeptide Hybrids InIn thisthis subsection,subsection, wewe mainlymainly highlightedhighlighted supramolecular architectures architectures comprising comprising covalently covalently conjugatedconjugated nucleicnucleic acid–oligopeptideacid–oligopeptide hybrids as theirtheir components.components. This This subsection subsection is is further further divideddivided into into two two subsubsections subsubsections (2.2.1: (Section spherical, 2.2.1 :2.2.2: spherical, fibrous) Section based on 2.2.2 the: primary fibrous) morphology based on theof primarythe supramolecular morphology architectures. of the supramolecular Generally, architectures. covalent conjugations Generally, covalentcause the conjugations self-assembly cause of the the self-assemblyresultant molecular of the resultanthybrids into molecular supramolecular hybrids into architectures supramolecular more architectures predictably morewhen predictablyeither the whennucleic either acid theor oligopeptide nucleic acid component or oligopeptide exhibits component robust ability exhibits to robustform specific ability tosupramolecular form specific supramoleculararchitectures. Indeed, architectures. most Indeed,of the mostfollowing of the followingrecent research recent research exploited exploited the conjugation the conjugation of ofself-assembling self-assembling peptides peptides onto onto the the nucleic nucleic acids acids of of interest, interest, thereby thereby producing producing novel novel supramolecular supramolecular architecturesarchitectures underunder givengiven aqueousaqueous conditions.

2.2.1. Spherical Supramolecular Architectures 2.2.1. Spherical Supramolecular Architectures SinceSince the discovery of self-assembly ability of of the the FF FF dipeptide dipeptide to to form form nanotubes nanotubes in in water water [78], [78], itit hashas beenbeen usedused asas aa promisingpromising motifmotif toto designdesign self-assemblingself-assembling molecules [79–90]. [79–90]. Thus, Thus, the the design design ofof aa covalentlycovalently conjugated hybrid containing the the FF FF dipeptide dipeptide and and an an oligo oligo DNA DNA (ssDNA: (ssDNA: 5 -CTCTCTCTCTTT-3 , 12 nt), that is, ssDNA12-FF (Figure 10A), was expected, followed by an 50′-CTCTCTCTCTTT-3′0, 12 nt), that is, ssDNA12-FF (Figure 10A), was expected, followed by an investigationinvestigation ofof thethe formationsformations ofof theirtheir correspondingcorresponding supramolecularsupramolecular architecturesarchitectures [[91].91]. Initial Initial assessmentsassessments by Vebert-Nardin and coworkers via microscopic observations (AFM (AFM and and scanning scanning electronelectron microscopy) microscopy) revealed revealed that that spherical spherical supram supramolecularolecular nanostructures nanostructures with withsizes sizesranging ranging from from 200 to 300 nm were successfully obtained by the direct dissolution of ssDNA -FF in water 200 to 300 nm were successfully obtained by the direct dissolution of ssDNA12-FF12 in water (2 (2mg/mL), mg/mL), followed followed by byextrusion extrusion through through a filter a filter membrane membrane (0.45 (0.45 μm).µ m).Such Such a morphology a morphology was wasnot notobserved observed by only by only mixing mixing the the unmodified unmodified FF FFdipeptide dipeptide with with the the component component oligo oligo DNA DNA (12 (12 nt) nt) withoutwithout covalentcovalent conjugation. conjugation. Furthermore, Furthermore, their thei hollowr hollow vesicular vesicular structures structures (Figure (Figure 10B) 10B) were were made evidentmade evident by detailed by detailed microscopic microscopic observations observat (TEMions and (TEM confocal and microscopy) confocal microscopy) and the encapsulation and the

Int. J. Mol. Sci. 2020, 21, 9458 12 of 25 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 25 abilityencapsulation of a hydrophilic ability of fluorescencea hydrophilic dye. fluorescenc Subsequently,e dye. Subsequently, the same research the same group research reported group that anotherreported aromatic that another dipeptide aromatic (the dipeptide tryptophan (the tryp (WW)tophan dipeptide), (WW) dipeptide), which conjugated which conjugated with the samewith oligo DNA (ssDNA: 5 -CTCTCTCTCTTT-3 , 12 nt), ssDNA12-WW (Figure 10C), yielded a spherical the same oligo DNA (ssDNA:0 5′-CTCTCTCTCTTT-30 ′, 12 nt), ssDNA12-WW (Figure 10C), yielded a supramolecularspherical supramolecular nanostructure nanostructure with a size with similar a size to similar that of to its that FF counterpartof its FF counterpart (described (described above) at aabove) lowered at concentration.a lowered concentration. Interestingly, Interestingly, at an increased at an concentration, increased concentration, e.g., 1.0 mM, e.g.,ssDNA 1.012 mM,-WW produced fibrous supramolecular nanostructures (the diameter ranged between 0.5 and 1.0 µm and ssDNA12-WW produced fibrous supramolecular nanostructures (the diameter ranged between 0.5 theand length 1.0 μm was and several the length micrometers), was several as micrometers), depicted in Figure as depicted 10D[92 in]. Figure 10D [92].

FigureFigure 10.10. ((AA)) ChemicalChemical structurestructure ofof ssDNAssDNA1212-FF-FF..( (B)) Plausible supramolecular supramolecular architectures architectures of of the the hollowhollow vesicularvesicular structures structures that that were were obtained obtained through through the self-assembly the self-assembly of ssDNA of12 ssDNA-FF.(C)12 Chemical-FF. (C) structureChemical ofstructuressDNA12 -WWof ssDNA.(D) Plausible,12-WW. ( concentration-dependentD) Plausible, concentration- supramoleculardependent supramolecular architectures of ssDNAarchitectures12-WW ofobtained ssDNA12 by-WW its self-assembly.obtained by its Adapted self-assembly. from[ 91Adapted,92]. Copyright from [91,92]. 2012 Copyright and 2014 Royal2012 Chemicaland 2014 Royal Society. Chemical Society.

SphericalSpherical capsular capsular architectures, architectures, which which displayed displayed nucleic nucleic acids acids at their at theirsurfaces surfaces (exterior), (exterior), were weresuccessfully successfully constructed constructed by Ma bytsuura Matsuura and andcoworkers coworkers [93] [ 93th]rough through the the self-assembly self-assembly of of a a DNADNA/peptide/peptide hybrid,hybrid,βAF-dA 20βAF-dA(Figure20 11A),(Figure comprising 11A), a peptide comp (INHVGGTGGAIMAPVAVTRrising a peptide QLVGS,(INHVGGTGGAIMAPVAVTRQLVGS, 24 aa) that was covalently conjugated 24 aa) that to the was nucleic covalently acid (ssDNA: conjugated A20, to 20 the nt). nucleic The 24-mer acid peptide(ssDNA: is A20, a β -annulus20 nt). The fragment 24-mer peptide (βAF), whichis a β-annulus is a structural fragment motif (βAF of), the which internal is a structural skeleton ofmotif the tomatoof the internal bushy stunt skeleton virus of capsid. the tomato The research bushy groupstunt virus demonstrated capsid. The that research the 24-mer groupβ-annulus demonstrated peptide fragmentthat the could24-mer self-assemble β-annulus intopeptide spherical fragment capsular could nanostructures self-assemble with into a sizespherical of 30–50 capsular nm in waternanostructures [94]. Similarly, with a βsizeAF-dA of 30–5020 could nm self-assemblein water [94]. intoSimilarly, spherical βAF-dA nanostructures,20 could self-assemble as depicted into in Figurespherical 11A, nanostructures, which was observed as depicted by TEM in andFigure dynamic 11A, which light scattering was observed (DLS) by measurements TEM and dynamic (98 63 light nm, ± thescattering average (DLS) hydrodynamic measurements diameter). (98 ± 63 Although nm, the the average DNA strandhydrodynamic (dA20) was diameter). attached Although to the 24-mer the βDNA-annulus strand peptide (dA20) fragment, was attachedβAF-dA to the20 could24-mer still β-annulus self-assemble peptide at lowerfragment, concentrations βAF-dA20 could compared still withself-assemble that of the at original lower concentrations DNA-unmodified compared peptide wi fragment.th that of the Similarly, original the DNA-unmodified spherical nanostructures peptide obtainedfragment. from Similarly,βAF-dT the20 (Figure spherical 11A) nanostructures with an average obtained hydrodynamic from β diameterAF-dT20 of(Figure 65 20 11A) nm (estimated with an ± byaverage DLS) werehydrodynamic constructed diameter under theof 65 same ± 20 conditions. nm (estimated Thereafter, by DLS) to were confirm constructed the hybridization under the abilitysame ofconditions. DNAs that Thereafter, were displayed to confirm on thethe sphericalhybridization nanostructures, ability of DNAs the two that spherical were displayed nanostructures on the werespherical mixed nanostructures, equimolarly. the The two TEM spherical observations nanostructures and DLS measurementswere mixed equimolarly. revealed the The presence TEM ofobservations their aggregates, and DLS which measurements were constructed revealed through the presence the hybridization of their ofaggregates, complementary which DNAswere constructed through the hybridization of complementary DNAs (dA20•dT20). Further experimental (dA20 dT20). Further experimental pieces of evidence for the DNA hybridization ability of the spherical pieces• of evidence for the DNA hybridization ability of the spherical nanostructures of βAF-dT20 were obtained by complexing βAF-dT20 with long complementary DNA strands. For example, the

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 25 nanostructures of βAF-dT20 were obtained by complexing βAF-dT20 with long complementary DNA strands. For example, the addition of polydA caused extensive aggregations of the βAF-dT20 spherical addition of polydA caused extensive aggregations of the βAF-dT20 spherical nanostructures, nanostructures,whereas no aggregation whereas nowas aggregation observed upon was observedadding polydT. upon adding Subsequently polydT., Subsequently,another DNA/peptide another DNA/peptide hybrid, βAF-ssDNA23 (ssDNA : TCTACAAAGGGAAGCCCTTTCTG; 23 nt) in which hybrid, βAF-ssDNA23 (ssDNA23: TCTACAAAGGGAAGCCCTTTCTG;23 23 nt) in which the ssDNA thestrands ssDNA were strands directed were inside directed the capsular inside thearchitec capsulartures, architectures,was designed was (Figure designed 11B). The (Figure obtained 11B). TheDNA-encapsulated obtained DNA-encapsulated spherical nanost sphericalructure nanostructure exhibited reduction-responsive exhibited reduction-responsive disassembly disassembly that was thatdue wasto the due reductive to the reductive cleavage cleavageof the disul of thefide disulfide bonds between bonds between DNA and DNA the and peptide, the peptide, which whicheventually eventually resulted resulted in the in controlled the controlled release release of the of the encapsulated encapsulated DNAs DNAs [95] [95.]. Overall, Overall, the the spherical β architectures of thethe 24-mer24-mer peptidepeptide β-annulus conjugates were susufficientlyfficiently robust [[96–103]96–103] soso thatthat variousvarious DNAsDNAs couldcould bebe introduced intointo the exterior and interior parts of the capsular structure since thethe introducedintroduced DNADNA diddid notnot destroydestroy thethe abilityability ofof thethe peptidespeptides toto formform thethe capsularcapsular structure.structure.

Figure 11.11. ((AA)) SchematicSchematic representation representation of of the the self-assembly self-assembly of βof-annulus β-annulus fragment fragment (βAF (βAF)_dN)_dN20 [N20 [N= A = (Aβ AF_dA(βAF_dA20)20 or) or T (βT AF_dT(βAF_dT20)]20 to)] formto form spherical spherical capsular capsular architectures architectures that displayedthat displayed the nucleic the nucleic acids atacids their at exteriortheir exte surfaces.rior surfaces. (B) Schematic (B) Schematic representation representation of the of self-assembly the self-assembly of βAF_ssDNA of βAF_ssDNA23 to form23 to sphericalform spherical capsular capsular architectures architectures that encapsulated that encapsulated the nucleic the acids. nucleic Adapted acids. fromAdapted ref 88. from Copyright ref 88. 2017Copyright John Wiley 2017 andJohn Sons Wiley Publisher. and Sons Adapted Publisher. from Adapted [95]. Copyright from [95]. 2019 Copyright the Chemical 2019 Society the Chemical of Japan. Society of Japan. Lim and a coworker designed two DNA/peptide hybrids, β-suRGD-AS and β-suRGD-S (FigureLim 12 ),and comprising a coworker ssDNAs designed (β-suRGD-AS two DNA/peptide: GCGAGCTGCACGCTGCCGTC, hybrids, β-suRGD-AS 20 and nt; ββ-suRGD-S-suRGD-S: GACGGCAGCGTGCAGCTC,(Figure 12), comprising ssDNAs 18 nt) and (β a-suRGD-ASβ-sheet peptide: GCGAGCTGCACGCTGCCGTC, (β-suRGD: KWKWEWYWKWEWKRG 20 nt; DRGD,β-suRGD-S 19 aa): containingGACGGCAGCGTGCAGCTC, a self-assembly motif 18 and nt) a cationicand segment.a β-sheet They peptide demonstrated (β-suRGD that: theKWKWEWYWKWEWKRGDRGD, simultaneous and orthogonal molecular 19 aa) containing self-assembly a self-assembly abilities of motif DNA and a the cationic peptide segment. moiety inThey the demonstrated hybrids (β-suRGD-AS that the simultaneousand β-suRGD-S and orthogonal) enabled the molecular construction self-assembly of well-defined abilities toroidalof DNA nanostructures.and the peptide More moiety specifically, in the hybrids the two (β DNA-suRGD-AS/peptide hybridsand β-suRGD-S (β-suRGD-AS) enabledand theβ-suRGD-S construction) were of mixedwell-defined in a 1:1 stoichiometrictoroidal nanostructures. ratio, whereas twoMore distinct specifically, protocols werethe appliedtwo DNA/peptide to control the assemblyhybrids pathways(β-suRGD-AS (pathway and β 1:-suRGD-S peptide assembly) were mixed to DNA in assembly,a 1:1 stoichiometric pathway 2: ratio, DNA whereas assembly two to peptidedistinct assembly),protocols were as outlined applied in Figureto control 12. Thethe twoassembly pathways pathways caused (pathway the formation 1: peptide of toroidal assembly nanostructures, to DNA asassembly, revealed pathway by cryoTEM 2: DNA (3D reconstructionassembly to peptide of a single assembly), particle) as (particle outlined size in= Figure9 nm, as12. estimated The two bypathways TEM, AFM,caused and the DLS), formation which wereof toroidal almost identicalnanostructures, (β-suRGD-AS as revealed/β-suRGD-S by cryoTEM). By contrast, (3D heterogeneousreconstruction of structures a single particle) were obtained (particle by size simple = 9 nm, dissolution as estimated (without by TEM, any AFM, protocol and to DLS), control which the assemblywere almost pathways) identical most (β probably-suRGD-AS because/β-suRGD-S of the formations). By contrast, of kinetically heterogeneous trapped structures structures [104 ,were105]. Interestingly,obtained by thesimple structural dissolution transition (without from the any monodispersed protocol to control toroidal the nanostructures assembly pathways) to the entangled most fibers,probably including because the of giant the toroidalformations structures, of kinetically could be trapped induced structures upon adding [104,105]. mRNA, Interestingly, which contains the a structural transition from the monodispersed toroidal nanostructures to the entangled fibers, including the giant toroidal structures, could be induced upon adding mRNA, which contains a

Int. J. Mol. Sci. 2020, 21, 9458 14 of 25 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 25 sequencesequence (20(20 nt)nt) thatthat isis complementary complementary to to DNA DNAAS AS, and, and this this should should be be related related to to the the antisense antisense eff ecteffect of theof the nanostructures nanostructures (β-suRGD-AS (β-suRGD-AS/β-suRGD-S/β-suRGD-S) demonstrated) demonstrated in in the the study. study.

Figure 12. SequencesSequences of of the the two two DNA/peptide DNA/peptide hybrids, hybrids, β-suRGD-ASβ-suRGD-AS andand β-suRGD-Sβ-suRGD-S, and, andthe two the twodistinct distinct protocols protocols that that controlled controlled their their assembly assembly pathway pathway to to yield yield the the monodispersed toroidaltoroidal nanostructures ((β-suRGD-AS/β-suRGD-S-suRGD-S).). Adapted from [[104].104]. Copyright Copyright 2016 2016 John Wiley andand Sons Publisher.Publisher. 2.2.2. Fibrous Supramolecular Architectures 2.2.2. Fibrous Supramolecular Architectures As described by several examples in Section 2.2.1, the morphology of supramolecular As described by several examples in Section 2.2.1, the morphology of supramolecular architectures that are obtained from covalently conjugated nucleic acid–oligopeptide hybrids is architectures that are obtained from covalently conjugated nucleic acid–oligopeptide hybrids is conditional. Fibrous supramolecular nanostructures could be preferentially obtained by strengthening conditional. Fibrous supramolecular nanostructures could be preferentially obtained by the self-assembly ability of peptides for the direct formation of one-dimensional structures. strengthening the self-assembly ability of peptides for the direct formation of one-dimensional Indeed, fibrous supramolecular nanostructures have been constructed more efficiently by attaching structures. Indeed, fibrous supramolecular nanostructures have been constructed more efficiently by ssDNA to fluorenylmethyloxycarbonyl (Fmoc)-FF, as shown in Figure 13A. The Fmoc group attaching ssDNA to fluorenylmethyloxycarbonyl (Fmoc)-FF, as shown in Figure 13A. The Fmoc (one of the most common protecting groups for amino acids) is currently widely accepted as group (one of the most common protecting groups for amino acids) is currently widely accepted as a a powerful self-assembly motif because of its ability to form one-dimensional supramolecular powerful self-assembly motif because of its ability to form one-dimensional supramolecular nanostructures [106–108]. Interestingly, Freeman and coworkers revealed that the morphology nanostructures [106–108]. Interestingly, Freeman and coworkers revealed that the morphology of of the fibrous supramolecular nanostructures strongly depended on the length of ssDNA [109]. the fibrous supramolecular nanostructures strongly depended on the length of ssDNA [109]. In In detail, Fmoc-FF-ssDNA19 (ssDNA19: CTCAGTGGACAGCCTTTTT; 19 nt) afforded twisted fibers detail, Fmoc-FF-ssDNA19 (ssDNA19: CTCAGTGGACAGCCTTTTT; 19 nt) afforded twisted fibers with average width and pitch of 20 and 150 nm, respectively, whereas Fmoc-FF-ssDNA46 (ssDNA46: with average width and pitch of 20 and 150 nm, respectively, whereas Fmoc-FF-ssDNA46 (ssDNA46: CAGTACAGTTTCGTCCAACGCTCCAGAACTGAGGCTGTCCACTGAG: 46 nt) afforded loosely CAGTACAGTTTCGTCCAACGCTCCAGAACTGAGGCTGTCCACTGAG: 46 nt) afforded loosely twisted but wider fibers compared with those of Fmoc-FF-ssDNA19 with average width and pitch of twisted but wider fibers compared with those of Fmoc-FF-ssDNA19 with average width and pitch of 44 and 630 nm, respectively. Under the investigated conditions, Fmoc-FF did not produce twisted 44 and 630 nm, respectively. Under the investigated conditions, Fmoc-FF did not produce twisted fibers, thus suggesting that helical structures and their propensity to form self-assembled structures fibers, thus suggesting that helical structures and their propensity to form self-assembled structures of Fmoc-FF-ssDNAs are dictated by the length of ssDNA. Additionally, they confirmed that the of Fmoc-FF-ssDNAs are dictated by the length of ssDNA. Additionally, they confirmed that the hybridization of the DNA segment in Fmoc-FF-ssDNA46 caused fiber bundling, which could be more hybridization of the DNA segment in Fmoc-FF-ssDNA46 caused fiber bundling, which could be clearly observed in the co-assembly of Fmoc-FF-ssDNA19 and Fmoc-FF-ss(as)DNA19 in which the more clearly observed in the co-assembly of Fmoc-FF-ssDNA19 and Fmoc-FF-ss(as)DNA19 in which complementary ssDNA (ss(as)DNA19) against ssDNA19 was introduced, as shown in Figure 13B,C. the complementary ssDNA (ss(as)DNA19) against ssDNA19 was introduced, as shown in Figure Accordingly, precise the base pairing (hybridization) ability of nucleic acids (DNA) was undoubtedly 13B,C. Accordingly, precise the base pairing (hybridization) ability of nucleic acids (DNA) was beneficial to the control of assembled structures at molecular and supramolecular levels, as highlighted undoubtedly beneficial to the control of assembled structures at molecular and supramolecular in this paper. In fact, Stupp and coworkers, including the same author (Freeman), reported earlier levels, as highlighted in this paper. In fact, Stupp and coworkers, including the same author that peptide amphiphiles, which contained nucleic acids, produced super-structured networks (Freeman), reported earlier that peptide amphiphiles, which contained nucleic acids, produced super-structured networks with controlled and reversible bundling of supramolecular fibrous

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 25 with controlled and reversible bundling of supramolecular fibrous structures [110]. In the study, morestructures crucially, [110]. the In bundling the study, of more the fibrous crucially, structures the bundling influenced of the the fibrous phenotypic structures transformations influenced the in thephenotypic astrocytes transformations (neural cells) thatin the were astrocytes in contact (neural with cells) the materials.that were in Conversely, contact with the the polyanionic materials. propertyConversely, of nucleicthe polyanionic acid can, property in turn, of aff nucleicord a means acid can, of controllingin turn, afford the a nucleation means of controlling of the peptide the self-assembly,nucleation of whichthe peptide might self-assembly, be involved in which polyanion-induced might be involved amyloid in fibrillationpolyanion-induced that is associated amyloid withfibrillation the corresponding that is associated diseases. with Forthe example,correspon Lynding and diseases. coworkers For example, recently succeededLyn and coworkers to obtain high-resolutionrecently succeeded structural to obtain information high-resolution on the self-assembled structural nanostructuresinformation on of athe positively self-assembled charged peptidenanostructures (Ac-KLVIIAG-NH of a positively2: 7 aa) charged that was peptide templated (Ac-KLVIIAG-NH by nucleic acids,2: 7 whichaa) that was was facilitated templated by theby structuralnucleic acids, complementarity which was facilitated between the by nucleic the structural acid backbone complementarity and the antiparallel between cross-the nucleicβ structures acid ofbackbone the peptides and the for antiparallel electrostatic cross- interactionsβ structures [111]. of the peptides for electrostatic interactions [111].

FigureFigure 13. 13.(A )(A Chemical) Chemical structure structure of Fmoc-FF-ssDNA of Fmoc-FF-ssDNAs. (B) Schematics. (B) Schematic representation representation of the self- assembly of the ofself-assemblyFmoc-FF-ssDNA of Fmoc-FF-ssDNAs to form hierarchicals to form nanostructures hierarchical through nanostruct DNAures hybridization. through DNA (C) Representativehybridization. TEM(C) imagesRepresentative obtained fromTEM co-assemblyimages obtained of Fmoc-FF-ssDNA from co-assembly19 and Fmoc-FF-ss(as)DNA of Fmoc-FF-ssDNA19. Scale19 and bar: 200Fmoc-FF-ss(as)DNA nm. Adapted from19. [109Scale]. Copyrightbar: 200 nm. 2019 Adapted American from Chemical [109]. Copyright Society. 2019 American Chemical Society. 3. Nucleic Acid/Polypeptide (Protein) Hybrids 3. NucleicIn this Acid/Polypeptide section, we briefly (Protein) highlighted Hybrids supramolecular architectures comprising nucleic acid/polypeptide(protein)In this section, we hybrids briefly as highlighted their components, supramolecular and the hybrids architectures are discussed comprising in two subsections nucleic basedacid/polypeptide(protein) on their types of conjugations. hybrids as their components, and the hybrids are discussed in two subsections based on their types of conjugations. 3.1. Non-Covalently Conjugated Hybrids 3.1. Non-covalentlyMayo and coworkers Conjugated designed Hybrids and constructed well-defined supramolecular nanowires comprisingMayo dsDNAand coworkers/artificially designed modified and protein construct (dualENHed well-defined) complexes supramolecular in which a sophisticated nanowires supramolecularcomprising dsDNA/artificially assembly was obtained modified via protein selective (dualENH protein–protein) complexes interaction in which (homodimerization) a sophisticated thatsupramolecular was aided by aassembly computational was design, obtained as well asvia by inherentselective protein protein–protein/dsDNA interactions interaction [112]. They(homodimerization) selected the well-studied that was Drosophilaaided byengrailed a computational homeodomain design, (ENH) as aswell the as sca ffbyold inherent protein. Homeodomains,protein/dsDNA includinginteractions ENH, [112]. are commonThey eukaryoticselected the DNA-binding well-studied domains Drosophila comprising engrailed three heliceshomeodomain (conventionally (ENH) definedas the asscaffold 60 aa inprotein. length basedHomeodomains, on homology) including [113]. The ENH, homodimerization are common interfaceeukaryotic of DNA-bindingdualENH was domains engineered comprising on the exteriorthree helices sides (conventionally of helices 1 and defined 2 of ENH as 60 that aa arein structurallylength based opposite on homology) its DNA-binding [113]. The homodimerization helix 3. To obtain interface a one-dimensional of dualENH was DNA engineered/dualENH on assembly,the exterior dsDNA sides of requires helices two 1 and protein-binding 2 of ENH that sites are structurally with the desired opposite geometry its DNA-binding (180◦). Specifically, helix 3. uponTo obtain complexing a one-dimensional dsDNA (25 bp) DNA/ containingdualENH an 11-nucleotideassembly, dsDNA binding requires motif (TAATTTAATTT,two protein-binding TAATTT: sites ENH-bindingwith the desired motif) geometry with dualENH (180 °). Specifically,, the nanowire upon structures complexing (width, dsDNA ~15 nm;(25 bp) length, containing ~300 nm;an as11-nucleotide estimated by binding AFM) weremoti successfullyf (TAATTTAATTT, obtained. TAATTT: Noteworthily, ENH-binding the crystal motif) structure with ofdualENH the complex, the wasnanowire also solved structures in which (width, a slightly ~15 kinkednm; length, (may not~300 reflect nm; theas estimated structure of by the AFM) nanowire were in successfully the solution state)obtained. but infinitelyNoteworthily, repeated the formationcrystal structure of the dsDNA of the /proteincomplex nanowires was also wassolved evident in which (Figure a slightly14). kinked (may not reflect the structure of the nanowire in the solution state) but infinitely repeated formation of the dsDNA/protein nanowires was evident (Figure 14).

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Figure 14. (A) Schematic representation of the formation of nanowires comprising a computationally designed protein/dualENH and a short dsDNA. (B) A representative AFM image obtained after FigureFigure 14.14. ((AA)) SchematicSchematic representationrepresentation ofof the formation of nanowires comprising a computationally computationally mixing dualENH with the dsDNA (25 bp). Adapted from [112]. Copyright 2014 Springer Nature. designeddesigned protein protein//dualENHdualENHand and a shorta short dsDNA. dsDNA. (B) A(B representative) A representative AFM AFM image image obtained obtained after mixing after dualENHmixing dualENHwith the with dsDNA the dsDNA (25 bp). (25 Adapted bp). Adapted from [112 from]. Copyright [112]. Copyright 2014 Springer 2014 Springer Nature. Nature. 3.2. Covalently Conjugated Hybrids 3.2.3.2. CovalentlyCovalently ConjugatedConjugated HybridsHybrids 3.2.1. Linear Supramolecular Architectures 3.2.1. Linear Supramolecular Architectures 3.2.1.Recently, Linear Supramolecular the hybridization Architectures chain reaction (HCR) of nucleic acids is recognized as a robust strategyRecently,Recently, for the constructingthe hybridization hybridization dsDNA-based chain chain reaction reaction (HCR)supr (HCR)amolecular of nucleic of nucleic acidsarchitectures acids is recognized is recognized for as aa robust asvariety a strategyrobust of bio-applicationsforstrategy constructing for constructing dsDNA-based[114,115]. As adsDNA-based supramolecular covalently conjugated architecturessupramolecular DNA/protein for a varietyarchitectures hybrid, of bio-applications Mirkin for anda variety coworkers [114, 115of]. Asconstructedbio-applications a covalently a set conjugated [114,115].of mutant As DNAGFPs a covalently/ bearingprotein hybrid,a haconjugatedirpin-shaped Mirkin DNA/protein and ssDNA coworkers and hybrid, demonstrated constructed Mirkin and a the set coworkers formation of mutant ofGFPsconstructed linear bearing supramolecular a aset hairpin-shaped of mutant oligomers GFPs ssDNA bearing (several and a ha demonstratedtensirpin-shaped of nanometers ssDNA the formation in and length, demonstrated of as linear estimated supramolecular the formationby AFM), whicholigomersof linear tethered supramolecular (several the tensprotein of oligomers nanometersthrough a (severalchain in length, growth tens as of polymerization estimated nanometers by AFM),in reaction length, which thatas estimated tetheredwas inducedthe by protein AFM),by the additionthroughwhich tethered aof chainan initiator the growth protein (ssDNA) polymerization through [116] a chain(Figure reaction growth 15). Notably, that polymerization was a induced further reaction extension by the that addition of was the inducedchain of an could initiator by the be (ssDNA)inducedaddition because [of116 an] (Figureinitiator the HCR15 (ssDNA)). Notably,system [116] aexhibited (Figure further 15). extensiona liviNotably,ng ofcharacter a the further chain that extension could was be similar of induced the chain to because the could living thebe polymerizationHCRinduced system because exhibited of covalentthe HCR a living polymers. system character exhibitedFurthermore, thatwas a livi similartheng same character to research the living that group polymerizationwas verysimilar recently to ofthe reported covalent living polymers.thatpolymerization the length Furthermore, ofof covalent the thelinear polymers. same supramolecular research Furthermore, group oligomers very the recently same can research reportedbe finely group that modulated thevery length recently by of thereportedcarefully linear designingsupramolecularthat the lengtha metastable oligomers of the hairpin-shapedlinear can be supramolecular finely modulated ssDNA oligomers(with by carefully the introductioncan designing be finely aof metastable modulatedmismatched hairpin-shaped by base carefully pairs) thatssDNAdesigning would (with abe metastable theattached introduction to hairpin-shaped the protein of mismatched [117]. ssDNA base (with pairs) the that introduction would be attached of mismatched to the protein base pairs) [117]. that would be attached to the protein [117].

Figure 15. SchematicSchematic representation representation of of the the formation of linear supramolecular architectures that tetheredFigure 15. the Schematic proteins bybyrepresentation exploitingexploiting hybridizationhybridizatio of the formationn chainchain of reactionreaction linear supramolecular (HCR)(HCR) ofof nucleicnucleic architectures acids.acids. Adapted that fromtethered [[116].116 ].the Copyright Copyright proteins 2018 by exploiting Amer Americanican hybridizatio Chemical Society. n chain reaction (HCR) of nucleic acids. Adapted from [116]. Copyright 2018 American Chemical Society. 3.2.2. Cage-Like Supramolecular Architectures 3.2.2. Cage-Like Supramolecular Architectures 3.2.2.Stephanopoulos Cage-Like Supramolecular and coworkers Architectures developed discrete three-dimensional cage-like supramolecular Stephanopoulos and coworkers developed discrete three-dimensional cage-like architectures by exploiting the specific self-assembly process of both the proteins and the nucleic supramolecularStephanopoulos architectures and bycoworkers exploiting thedeveloped specific self-assemblydiscrete three-dimensional process of both thecage-like proteins acids [118]. A homotrimeric protein containing three ssDNAs was constructed through the andsupramolecular the nucleic architecturesacids [118]. Aby homotrimeric exploiting the pr specoteinific containing self-assembly three process ssDNAs of bothwas theconstructed proteins covalent conjugation of ssDNAs to a cysteine residue (introduced by site-directed mutagenesis) throughand the thenucleic covalent acids conjugation[118]. A homotrimeric of ssDNAs prtootein a cysteine containing residue three (introduced ssDNAs wasby site-directedconstructed of 2-dehydro-3-deoxyphosphogluconate aldolase (25 kDa, C3-symmetric homotrimer) (Figure 16). mutagenesis)through the covalentof 2-dehydro-3-deoxyph conjugation of osphogluconatessDNAs to a cysteine aldolase residue (25 kDa, (introduced C3-symmetric by homotrimer)site-directed This covalently conjugated DNA/protein hybrid can form a discrete three-dimensional cage-like (Figuremutagenesis) 16). This of 2-dehydro-3-deoxyphcovalently conjugatedosphogluconate DNA/protein hybrid aldolase can (25 form kDa, a Cdiscrete3-symmetric three-dimensional homotrimer) supramolecular architecture by complexing with a triangular DNA nanostructure bearing three cage-like(Figure 16). supramolecular This covalently architecture conjugated by DNA/protei complexingn withhybrid a triangularcan form aDNA discrete nanostructure three-dimensional bearing complementary ssDNAs through DNA hybridization (Figure 16). The size of the three-dimensional threecage-like complementary supramolecular ssDNAs architecture through by complexing DNA hybridization with a triangular (Figure DNA 16). nanostructure The size bearingof the cage-like supramolecular architecture can be varied by changing the lengths of DNAs. Indeed, three-dimensionalthree complementary cage-like ssDNAs supramolecular through DNAarchitec hybridizationture can be varied (Figure by changing16). The thesize lengths of the of two different sizes of the supramolecular architectures (triangular pyramid: 8 nm (height) 10 nm DNAs.three-dimensional Indeed, two cage-like different supramolecular sizes of the supramol architececularture can architectures be varied by(triangular changing pyramid: the lengths× 8 nm of (side of the triangle), 12 nm (height) 14 nm (side of the triangle)) were constructed and visualized (height)DNAs. Indeed,× 10 nm two (side different of the sizes triangle), of× the supramol12 nm (height)ecular architectures× 14 nm (side (triangular of the pyramid:triangle)) 8were nm via AFM. constructed(height) × 10and nm visualized (side of via the AFM. triangle), 12 nm (height) × 14 nm (side of the triangle)) were constructed and visualized via AFM.

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Figure 16. Three-dimensional cage-likecage-like supramolecularsupramolecular architecturearchitecture obtained obtained by by the the complexation complexation of of a homotrimerica homotrimeric protein protein containing containing three three ssDNAs ssDNAs with witha atriangular triangular DNADNA nanostructurenanostructure bearing three complementary ssDNAsssDNAs through through DNA DNA hybridization. hybridizat Adaptedion. Adapted from [118from]. Copyright [118]. Copyright 2019 American 2019 ChemicalAmerican Society.Chemical Society.

3.2.3. Crystalline Supramolecular Architectures Tezcan andand coworkers coworkers designed designed and constructedand construc discreteted crystallinediscrete nucleoproteincrystalline nucleoprotein architectures througharchitectures the threethrough distinct the three cooperative distinct interactions: cooperative (i)interactions: the WC base (i) pairing,the WC (ii)base the pairing, DNA/ protein(ii) the interactions,DNA/protein and interactions, (iii) the protein–metal and (iii) the coordination protein–me [tal119 ].coordination Figure 17 shows [119]. the Figure molecular 17 shows design the of themolecular nucleoprotein design (covalentlyof the nucleoprotein conjugated (covalently protein/nucleic conjugated acid hybrid). protein/nucleic Therein, a acid modified hybrid). cytochrome, Therein, RIDC3a modified(an cytochrome, engineered RIDC3 variant (an of theengineered monomeric variant four-helix of the monomeric bundle protein four-helix cytochrome, bundle proteincb562), which was developed by the same group [120–122], was used as the protein component. Complementary cytochrome, cb562), which was developed by the same group [120–122], was used as the protein ssDNAscomponent. (TTATTAAAA Complementary and TTTTAATTAA ssDNAs (TTATTAAAA for RIDC3-10a andand TTTTAATTAARIDC3-10b, respectively, for RIDC3-10a 10 nt) wereand attachedRIDC3-10b to, RIDC3respectively,by exploiting 10 nt) were a single attached cysteine to RIDC3 residue by exploiting of RIDC3 .a single Essentially, cysteineRIDC3 residueitself of 2+ canRIDC3 self-assemble. Essentially, into RIDC3 one-, two-, itself and can three-dimensional self-assemble into crystalline one-, arraystwo-, throughand three-dimensional Zn -mediated interactionscrystalline arrays (protein–metal through Zn coordination).2+-mediated interactions As shown in (protein–metal Figure 17, ordered coordination). crystalline As architectures shown in (thin,Figure micrometer-sized 17, ordered crystalline crystals) architectures were obtained (thin, from micrometer-sizedRIDC3-10a and RIDC3-10bcrystals) were, although obtained under from a ratherRIDC3-10a small and window RIDC3-10b of conditions, although {pH (4.75–5),under a temperaturerather small (4–10window◦C), of and conditions stoichiometry {pH between(4.75–5), 2+ Zntemperatureand RIDC3-10a (4–10 °C),/10b and(2–10 stoichiometry equiv.)}. For example,between theZn solutions2+ and RIDC3-10a of RIDC3-10a/10b/ 10b(2–10did equiv.)}. not produce For 2+ anyexample, discretely the solutions assembled of architecture RIDC3-10a (crystals)/10b did innot the produce absence any of Zndiscretely. Furthermore, assembled the architecture addition of ethylenediaminetetraacetic(crystals) in the absence of acidZn2+. to Furthermore, the suspension the of addition the RIDC3-10a of ethy/lenediaminetetraacetic10b crystals dissolved it, acid as well to the as 2+ itssuspension incubation of at the>40 RIDC3-10a◦C. These results/10b crystals implied dissolved that Zn -it, and as DNA well hybridization-mediatedas its incubation at >40 interactions °C. These areresults keys implied to the formation that Zn2+- of and the DNA ordered hybridization-mediated crystalline architectures. interactions Furthermore, are keys by combining to the formation several techniquesof the ordered (negative crystalline stain TEM, architectures. cryoTEM, Furthermore, SAXS, and molecular by combining dynamics several simulations), techniques the structural(negative characterizationstain TEM, cryoTEM, of the SAXS, ordered and crystalline molecular architectures dynamics wassimulations), comprehensively the structural accomplished. characterization of the ordered crystalline architectures was comprehensively accomplished.

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FigureFigure 17.17. ((AA)) MolecularMolecular designdesign ofof thethe nucleoproteinsnucleoproteins ((RIDC3-10a and RIDC3-10b). ( (B)) Schematic representationrepresentation andand ((CC)) aa representativerepresentative TEMTEM imageimage of thethe orderedordered crystalline architectures obtained obtained 2+ throughthrough the the complexation complexation of ofRIDC3-10a RIDC3-10a/10b/10bwith with Zn Zn2+ under under a smalla small window window of conditions.of conditions. Scale Scale bar: 5bar:µm. 5 Adaptedμm. Adapted from from [119]. [119]. Copyright Copyrigh 2018t 2018 American American Chemical Chemical Society. Society.

4.4. ConclusionsConclusions OneOne challengechallenge forfor thisthis researchresearch fieldfield isis thethe hierarchicalhierarchical combination of plural supramolecular architecturesarchitectures obtained obtained from from not not only only self-assembling self-assembling peptides peptides but but also also self-assembling self-assembling nucleic nucleic acids acids at meso-scaleat meso-scale as as described described in in Section Section 2.1.3 2.1.3.. Such Such examples examples are are stillstill rarerare andand oftenoften require a multi-step andand tedioustedious process.process. Nevertheless, careful careful mole molecularcular design design enabling enabling the the orthogonal molecular self-assemblingself-assembling couldcould allowallow forfor simplesimple one-pot production process to obtain unique hierarchical supramolecularsupramolecular architecturesarchitectures [76 [76,77].,77]. Another Another simple simple but importantbut important challenge challenge is cost issue.is cost Although issue. reliableAlthough and reliable reproducible and reproducible automatic synthesis automatic of nucleicsynthesis acids of andnucleic peptides acids areand available, peptides theare production available, costthe issueproduction remains, cost especially issue remains, for DNA especially origami requiringfor DNA overorigami a hundred requiring ssDNAs over witha hundred different ssDNAs length andwith sequence different [ 123length] Biotechnological and sequence mass [123] production Biotechnological might be mass optional, production whereas might it poses be additionaloptional, issueswhereas such it poses as sterilization additional and issues batch-to-batch such as sterilization variability, and which batch-to-batch could result variability, in the increase which could in the productionresult in the costs. increase It might in the be worthproduction noting costs. thatcatalytic It might peptide be worth synthesis noting has that attracted catalytic increasing peptide attentionssynthesis recentlyhas attracted [124–128 increasi], whichng could attentions overcome recently the production [124–128], cost which issue ofcould peptides. overcome the productionIn a natural cost issue system, of peptides. the ribosome, which consists of RNA and polypeptides, i.e., ribosomal RNA (rRNA)In anda natural proteins, system, respectively the ribosome, [129–132 which], is one consists of the most of RNA complex and hierarchical polypeptides, bio-supramolecular i.e., ribosomal architecturesRNA (rRNA) and and exhibits proteins, a precise respectively function [129–132], to synthesize is one proteins of the (decodingmost complex center hierarchical or factory). Thisbio-supramolecular hybrid bio-supramolecular architectures architecture and exhibits clearly a precise reflects function a strong to structure–functionsynthesize proteins relationship (decoding throughcenter or the factory). construction This hybrid of evolutional bio-supramolecular but sophisticated architecture hybrid clearly architectures. reflects a Instrong sharp structure– contrast, thefunction formations relationship of rather through structureless the construction liquid droplets of evolutional in living but cells, sophisticated namely, the hybrid liquid–liquid architectures. phase separation,In sharp contrast, have very the recentlyformations attracted of rather increased structureless attention liquid in the droplets fundamental in living and cells, applied namely, research the fieldsliquid–liquid of biology, phase aswell separation, as chemistry have very [133– recently135]. Recent attracted studies increased have revealed attention that in the dynamicfundamental and transientand applied formation research of fields nucleic of acidbiology,/protein as well droplets as chemistry in the cells [133–135]. is closely Recent related studies to crucial have biological revealed processes,that the dynamic such as celland polarizationtransient formation [136–139 of]. nucleic These findings acid/protein could droplets significantly in the a ffcellsect theis closely design principlesrelated to ofcrucial molecular biological hybrid processes, materials insuch the as future. cell polarization [136–139]. These findings could significantlyConclusively, affect thisthe design review principles has described of molecular a variety hybrid of supramolecular materials in the architecturesfuture. that were assembledConclusively, from both this nucleic review acids has and described peptides a with variet structuraly of supramolecular orders that ranged architectures from the that molecular were toassembled nano-scales. from The both rational nucleic and acids modular and molecularpeptides andwith structural structural designs orders forthat the ranged construction from andthe engineeringmolecular to of suchnano-scales. supramolecular The rational architectures and mo todular equip molecular them with and desired structural functions designs and properties for the wouldconstruction facilitate and the engineering elucidation ofof theirsuch beneficial supramolec bio-applications,ular architectures such to as eq sensoruip them [140], with cell-culturing desired matrixfunctions for and regenerative properties medicine would facilitate [141–143 the], elucidation and drug-releasing of their beneficial material bio-applications, [144–147]. Furthermore, such as synergisticallysensor [140], cell-culturing combining thematrix strengths for regenerative of both molecules medicine (nucleic[141–143], acids and and drug-releasing peptides) would material be essential[144–147]. to Furthermore, widening the synergistically scope of future combining research projects. the strengths of both molecules (nucleic acids and peptides) would be essential to widening the scope of future research projects. Author Contributions: S.L.H., N.R., S.A.H., M.I. wrote the paper. All authors have read and agreed to the publishedAuthor Contributions: version of the manuscript.S.L.H., N.R., S.A.H., M.I. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Int. J. Mol. Sci. 2020, 21, 9458 19 of 25

Funding: This research was in part funded by Suzuken Foundation of Life Science and KOSÉ cosmetology research foundation And The APC was funded by Suzuken Foundation of Life Science. Acknowledgments: M.I. would like to thank financial supports from Suzuken Foundation of Life Science and KOSÉ cosmetology research foundation. S.L.H. thanks JSPS Research Fellowships for Young Scientists. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Seeman, N.C.; Sleiman, H.F. DNA nanotechnology. Nat. Rev. Mater. 2018, 3, 17068. [CrossRef] 2. Madsen, M.; Gothelf, K.V. Chemistries for DNA nanotechnology. Chem. Rev. 2019, 119, 6384–6458. [CrossRef] [PubMed] 3. Heuer-Jungemann, A.; Liedl, T. From DNA tiles to functional DNA materials. Trends Chem. 2019, 1, 799–814. [CrossRef] 4. Seeman, N.C. DNA in a material world. Nature 2003, 421, 427–431. [CrossRef] 5. Seeman, N.C. Nucleic acid junctions and lattices. J. Theor. Biol. 1982, 99, 237–247. [CrossRef] 6. Rothemund, P.W.K. Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440, 297–302. [CrossRef] 7. Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA origami: Scaffolds for creating higher order structures. Chem. Rev. 2017, 117, 12584–12640. [CrossRef] 8. Kallenbach, N.R.; Ma, R.-I.; Seeman, N.C. An immobile nucleic acid junction constructed from oligonucleotides. Nature 1983, 305, 829–831. [CrossRef] 9. Fu, T.J.; Seeman, N.C. DNA double-crossover molecules. Biochemistry 1993, 32, 3211–3220. [CrossRef] 10. Wang, Y.; Mueller, J.E.; Kemper, B.; Seeman, N.C. Assembly and characterization of five-arm and six-arm DNA branched junctions. Biochemistry 1991, 30, 5667–5674. [CrossRef] 11. Wang, X.; Chandrasekaran, A.R.; Shen, Z.; Ohayon, Y.P.; Wang, T.; Kizer, M.E.; Sha, R.; Mao, C.; Yan, H.; Zhang, X.; et al. Paranemic crossover DNA: There and back again. Chem. Rev. 2018, 119, 6273–6289. [CrossRef][PubMed] 12. Winfree, E.; Liu, F.; Wenzler, L.A.; Seeman, N.C. Design and self-assembly of two-dimensional DNA crystals. Nature 1998, 394, 539–544. [CrossRef][PubMed] 13. Yan, H. DNA-templated self-assembly of protein arrays and highly conductive nanowires. Science 2003, 301, 1882–1884. [CrossRef][PubMed] 14. Schulman, R.; Winfree, E. Synthesis of crystals with a programmable kinetic barrier to nucleation. Proc. Natl. Acad. Sci. USA 2007, 104, 15236–15241. [CrossRef][PubMed] 15. Rothemund, P.W.K.; Ekani-Nkodo, A.; Papadakis, N.; Kumar, A.; Fygenson, D.K.; Winfree, E. Design and characterization of programmable DNA nanotubes. J. Am. Chem. Soc. 2004, 126, 16344–16352. [CrossRef] 16. Yin, P.; Hariadi, R.F.; Sahu, S.; Choi, H.M.T.; Park, S.H.; LaBean, T.H.; Reif, J.H. Programming DNA tube circumferences. Science 2008, 321, 824–826. [CrossRef] 17. Liu, H.; Chen, Y.; He, Y.; Ribbe, A.E.; Mao, C. Approaching the limit: Can one DNA oligonucleotide assemble into large nanostructures? Angew. Chem. Int. Ed. 2006, 45, 1942–1945. [CrossRef] 18. Wilner, O.I.; Orbach, R.; Henning, A.; Teller, C.; Yehezkeli, O.; Mertig, M.; Harries, D.; Willner, I. Self-assembly of DNA nanotubes with controllable diameters. Nat. Commun. 2011, 2, 540. [CrossRef] 19. Zhang, D.Y.; Hariadi, R.F.; Choi, H.M.T.; Winfree, E. Integrating DNA strand-displacement circuitry with DNA tile self-assembly. Nat. Commun. 2013, 4, 1965. [CrossRef] 20. Liu, X.; Zhao, Y.; Liu, P.; Wang, L.; Lin, J.; Fan, C. Biomimetic DNA nanotubes: Nanoscale channel design and applications. Angew. Chem. Int. Ed. 2019, 58, 8996–9011. [CrossRef] 21. He, Y.; Ye, T.; Su, M.; Zhang, C.; Ribbe, A.E.; Jiang, W.; Mao, C. Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 2008, 452, 198–201. [CrossRef][PubMed] 22. Zhang, C.; He, Y.; Su, M.; Ko, S.H.; Ye, T.; Leng, Y.; Sun, X.; Ribbe, A.E.; Jiang, W.; Mao, C. DNA self-assembly: From 2D to 3D. Faraday Discuss. 2009, 143, 221. [CrossRef][PubMed] 23. Tian, C.; Li, X.; Liu, Z.; Jiang, W.; Wang, G.; Mao, C. Directed self-assembly of DNA tiles into complex nanocages. Angew. Chem. Int. Ed. 2014, 53, 8041–8044. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 9458 20 of 25

24. Fu, J.; Yang, Y.R.; Johnson-Buck, A.; Liu, M.; Liu, Y.; Walter, N.G.; Woodbury, N.W.; Yan, H. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 2014, 9, 531–536. [CrossRef][PubMed] 25. Linko, V.; Eerikäinen, M.; Kostiainen, M.A. A modular DNA origami-based enzyme cascade nanoreactor. Chem. Commun. 2015, 51, 5351–5354. [CrossRef] 26. Yu, Y.; Jin, B.; Li, Y.; Deng, Z. Stimuli-responsive DNA self-assembly: From principles to applications. Chem. Eur. J. 2019, 25, 9785–9798. [CrossRef] 27. Xiao, M.; Lai, W.; Man, T.; Chang, B.; Li, L.; Chandrasekaran, A.R.; Pei, H. Rationally engineered nucleic acid architectures for biosensing applications. Chem. Rev. 2019, 119, 11631–11717. [CrossRef] 28. Douglas, S.M.; Bachelet, I.; Church, G.M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 2012, 335, 831–834. [CrossRef] 29. Li, S.; Jiang, Q.; Liu, S.; Zhang, Y.; Tian, Y.; Song, C.; Wang, J.; Zou, Y.; Anderson, G.J.; Han, J.-Y.; et al. A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo. Nat. Biotechnol. 2018, 36, 258–264. [CrossRef] 30. Kopperger, E.; List, J.; Madhira, S.; Rothfischer, F.; Lamb, D.C.; Simmel, F.C. A self-assembled nanoscale robotic arm controlled by electric fields. Science 2018, 359, 296–301. [CrossRef] 31. Nummelin, S.; Shen, B.; Piskunen, P.; Liu, Q.; Kostiainen, M.A.; Linko, V. Robotic DNA nanostructures. ACS Synth. Biol. 2020, 9, 1923–1940. [CrossRef][PubMed] 32. Mei, Q.; Wei, X.; Su, F.; Liu, Y.; Youngbull, C.; Johnson, R.; Lindsay, S.; Yan, H.; Meldrum, D. Stability of DNA origami nanoarrays in cell lysate. Nano Lett. 2011, 11, 1477–1482. [CrossRef][PubMed] 33. Hahn, J.; Wickham, S.F.J.; Shih, W.M.; Perrault, S.D. Addressing the instability of DNA nanostructures in tissue culture. ACS Nano 2014, 8, 8765–8775. [CrossRef][PubMed] 34. Kielar, C.; Xin, Y.; Shen, B.; Kostiainen, M.A.; Grundmeier, G.; Linko, V.; Keller, A. On the stability of DNA origami nanostructures in low-magnesium buffers. Angew. Chem. Int. Ed. 2018, 57, 9470–9474. [CrossRef] 35. Mikkilä, J.; Eskelinen, A.-P.; Niemelä, E.H.; Linko, V.; Frilander, M.J.; Törmä, P.; Kostiainen, M.A. Virus- encapsulated DNA origami nanostructures for cellular delivery. Nano Lett. 2014, 14, 2196–2200. [CrossRef] 36. Auvinen, H.; Zhang, H.; Nonappa; Kopilow, A.; Niemelä, E.H.; Nummelin, S.; Correia, A.; Santos, H.A.; Linko, V.; Kostiainen, M.A. Protein coating of DNA nanostructures for enhanced stability and immunocompatibility. Adv. Healthc. Mater. 2017, 6, 1700692. [CrossRef] 37. Agarwal, N.P.; Matthies, M.; Gür, F.N.; Osada, K.; Schmidt, T.L. Block copolymer micellization as a protection strategy for DNA origami. Angew. Chem. Int. Ed. 2017, 56, 5460–5464. [CrossRef] 38. Ponnuswamy, N.; Bastings, M.M.C.; Nathwani, B.; Ryu, J.H.; Chou, L.Y.T.; Vinther, M.; Li, W.A.; Anastassacos, F.M.; Mooney, D.J.; Shih, W.M. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation. Nat. Commun. 2017, 8, 15654. [CrossRef] 39. Ahmadi, Y.; Llano, E.D.; Bariši´c,I. (Poly)cation-induced protection of conventional and wireframe DNA origami nanostructures. Nanoscale 2018, 10, 7494–7504. [CrossRef] 40. Wang, S.-T.; Gray, M.A.; Xuan, S.; Lin, Y.; Byrnes, J.; Nguyen, A.I.; Todorova, N.; Stevens, M.M.; Bertozzi, C.R.; Zuckermann, R.N.; et al. DNA origami protection and molecular interfacing through engineered sequence-defined peptoids. Proc. Natl. Acad. Sci. USA 2020, 117, 6339–6348. [CrossRef] 41. Dobson, C.M. Protein folding and misfolding. Nature 2003, 426, 884–890. [CrossRef][PubMed] 42. Riek, R.; Eisenberg, D.S. The activities of amyloids from a structural perspective. Nature 2016, 539, 227–235. [CrossRef][PubMed] 43. Lou, S.; Wang, X.; Yu, Z.; Shi, L. Peptide tectonics: Encoded structural complementarity dictates programmable self-assembly. Adv. Sci. 2019, 6, 1802043. [CrossRef][PubMed] 44. Fletcher, D.A.; Mullins, R.D. Cell mechanics and the cytoskeleton. Nature 2010, 463, 485–492. [CrossRef] [PubMed] 45. Ke, P.C.; Zhou, R.; Serpell, L.C.; Riek, R.; Knowles, T.P.J.; Lashuel, H.A.; Gazit, E.; Hamley, I.W.; Davis, T.P.; Fändrich, M.; et al. Half a century of amyloids: Past, present and future. Chem. Soc. Rev. 2020, 49, 5473–5509. [CrossRef][PubMed] 46. Knowles, T.P.J.; Vendruscolo, M.; Dobson, C.M. The amyloid state and its association with protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014, 15, 384–396. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 9458 21 of 25

47. Gremer, L.; Schölzel, D.; Schenk, C.; Reinartz, E.; Labahn, J.; Ravelli, R.B.G.; Tusche, M.; Lopez-Iglesias, C.; Hoyer, W.; Heise, H.; et al. Fibril structure of amyloid-β(1–42) by cryo–electron microscopy. Science 2017, 358, 116–119. [CrossRef] 48. Tayeb-Fligelman, E.; Tabachnikov, O.; Moshe, A.; Goldshmidt-Tran, O.; Sawaya, M.R.; Coquelle, N.; Colletier, J.-P.; Landau, M. The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril. Science 2017, 355, 831–833. [CrossRef] 49. Engelberg, Y.; Landau, M. The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure. Nat. Commun. 2020, 11, 3894. [CrossRef] 50. Channon, K.; Bromley,E.H.C.; Woolfson,D.N. Synthetic biology through biomolecular design and engineering. Curr. Opin. Struct. Biol. 2008, 18, 491–498. [CrossRef] 51. Robson Marsden, H.; Kros, A. Self-assembly of coiled coils in synthetic biology: Inspiration and progress. Angew. Chem. Int. Ed. 2010, 49, 2988–3005. [CrossRef][PubMed] 52. Fletcher, J.M.; Boyle, A.L.; Bruning, M.; Bartlett, G.J.; Vincent, T.L.; Zaccai, N.R.; Armstrong, C.T.; Bromley, E.H.C.; Booth, P.J.; Brady, R.L.; et al. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology. ACS Synth. Biol. 2012, 1, 240–250. [CrossRef] [PubMed] 53. Roy, S.; Caruthers, M. Synthesis of DNA/RNA and their analogs via phosphoramidite and H-phosphonate chemistries. Molecules 2013, 18, 14268–14284. [CrossRef][PubMed] 54. Hao, M.; Qiao, J.; Qi, H. Current and emerging methods for the synthesis of single-stranded DNA. Genes 2020, 11, 116. [CrossRef][PubMed] 55. Merrifield, R.B. Solid phase peptide synthesis. I. the synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149–2154. [CrossRef] 56. Mäde, V.; Els-Heindl, S.; Beck-Sickinger, A.G. Automated solid-phase peptide synthesis to obtain therapeutic peptides. Beilstein J. Org. Chem. 2014, 10, 1197–1212. [CrossRef] 57. Moss, G.P.; Smith, P.A.S.; Tavernier, D. Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC Recommendations 1995). Pure Appl. Chem. 1995, 67, 1307–1375. [CrossRef] 58. Ni, R.; Chau, Y. Structural mimics of viruses through peptide/DNA co-assembly. J. Am. Chem. Soc. 2014, 136, 17902–17905. [CrossRef] 59. Ni, R.; Chau, Y. Tuning the inter-nanofibril interaction to regulate the morphology and function of peptide/DNA co-assembled viral mimics. Angew. Chem. Int. Ed. 2017, 56, 9356–9360. [CrossRef] 60. Ni, R.; Chau, Y. Nanoassembly of oligopeptides and DNA mimics the sequential disassembly of a spherical virus. Angew. Chem. Int. Ed. 2020, 59, 3578–3584. [CrossRef] 61. Lindsey, J.S. Self-assembly in synthetic routes to molecular devices. biological principles and chemical perspectives. New J. Chem. 1991, 15, 153–180. 62. Philp, D.; Stoddart, J.F. Self-assembly in natural and unnatural systems. Angew. Chem. Int. Ed. 1996, 35, 1154–1196. [CrossRef] 63. Klug, A. The tobacco mosaic virus particle: Structure and assembly. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999, 354, 531–535. [CrossRef] 64. Zhou, K.; Ke, Y.; Wang, Q. Selective in situ assembly of viral protein onto DNA origami. J. Am. Chem. Soc. 2018, 140, 8074–8077. [CrossRef][PubMed] 65. Zhou, K.; Zhou, Y.; Pan, V.; Wang, Q.; Ke, Y. Programming dynamic assembly of viral proteins with DNA origami. J. Am. Chem. Soc. 2020, 142, 5929–5932. [CrossRef][PubMed] 66. Ruff, Y.; Moyer, T.; Newcomb, C.J.; Demeler, B.; Stupp, S.I. Precision templating with DNA of a virus-like particle with peptide nanostructures. J. Am. Chem. Soc. 2013, 135, 6211–6219. [CrossRef] 67. Kang, Z.; Meng, Q.; Liu, K. Peptide-based gene delivery vectors. J. Mater. Chem. B 2019, 7, 1824–1841. [CrossRef] 68. Jiang, T.; Meyer, T.A.; Modlin, C.; Zuo, X.; Conticello, V.P.; Ke, Y. Structurally ordered nanowire formation from co-assembly of DNA origami and collagen-mimetic peptides. J. Am. Chem. Soc. 2017, 139, 14025–14028. [CrossRef] 69. Jin, J.; Baker, E.G.; Wood, C.W.; Bath, J.; Woolfson, D.N.; Turberfield, A.J. Peptide assembly directed and quantified using megadalton DNA nanostructures. ACS Nano 2019, 13, 9927–9935. [CrossRef] 70. Buchberger, A.; Simmons, C.R.; Fahmi, N.E.; Freeman, R.; Stephanopoulos, N. Hierarchical assembly of nucleic acid/coiled-coil peptide nanostructures. J. Am. Chem. Soc. 2020, 142, 1406–1416. [CrossRef] Int. J. Mol. Sci. 2020, 21, 9458 22 of 25

71. Brizard, A.M.; van Esch, J.H. Self-assembly approaches for the construction of cell architecture mimics. Soft Matter 2009, 5, 1320–1327. [CrossRef] 72. Safont-Sempere, M.M.; Fernández, G.; Würthner, F. Self-sorting phenomena in complex supramolecular systems. Chem. Rev. 2011, 111, 5784–5814. [CrossRef][PubMed] 73. Draper, E.R.; Adams, D.J. How should multicomponent supramolecular gels be characterised? Chem. Soc. Rev. 2018, 47, 3395–3405. [CrossRef][PubMed] 74. Kubota, R.; Nakamura, K.; Torigoe, S.; Hamachi, I. The power of confocal laser scanning microscopy in supramolecular chemistry: In situ real-time imaging of stimuli-responsive multicomponent supramolecular hydrogels. ChemistryOpen 2020, 9, 67–79. [CrossRef] 75. Udomprasert, A.; Bongiovanni, M.N.; Sha, R.; Sherman, W.B.; Wang, T.; Arora, P.S.; Canary, J.W.; Gras, S.L.; Seeman, N.C. Amyloid fibrils nucleated and organized by DNA origami constructions. Nat. Nanotechnol. 2014, 9, 537–541. [CrossRef] 76. Higashi, S.L.; Shibata, A.; Kitamura, Y.; Hirosawa, K.M.; Suzuki, K.G.N.; Matsuura, K.; Ikeda, M. Hybrid soft nanomaterials composed of DNA microspheres and supramolecular nanostructures of semi-artificial glycopeptides. Chem. Eur. J. 2019, 25, 11955–11962. [CrossRef] 77. Higashi, S.L.; Hirosawa, K.M.; Suzuki, K.G.N.; Matsuura, K.; Ikeda, M. One-pot construction of multicomponent supramolecular materials comprising self-sorted supramolecular architectures of DNA and semi-artificial glycopeptides. ACS Appl. Bio Mater. 2020.[CrossRef] 78. Reches, M.; Gazit, E. Casting metal nanowires within discrete self-assembled peptide nanotubes. Science 2003, 300, 625–627. [CrossRef] 79. Görbitz, C.H. Nanotube formation by hydrophobic dipeptides. Chem. Eur. J. 2001, 7, 5153–5159. [CrossRef] 80. Du, X.; Zhou, J.; Xu, B. Supramolecular hydrogels made of basic biological building blocks. Chem. Asian J. 2014, 9, 1446–1472. [CrossRef] 81. Adler-Abramovich, L.; Gazit, E. The physical properties of supramolecular peptide assemblies: From building block association to technological applications. Chem. Soc. Rev. 2014, 43, 6881–6893. [CrossRef][PubMed] 82. Fleming, S.; Ulijn, R.V. Design of nanostructures based on aromatic peptide amphiphiles. Chem. Soc. Rev. 2014, 43, 8150–8177. [CrossRef][PubMed] 83. Tao, K.; Makam, P.; Aizen, R.; Gazit, E. Self-assembling peptide semiconductors. Science 2017, 358.[CrossRef] [PubMed] 84. Tsuzuki, T.; Kabumoto, M.; Arakawa, H.; Ikeda, M. The effect of carbohydrate structures on the hydrogelation ability and morphology of self-assembled structures of peptide–carbohydrate conjugates in water. Org. Biomol. Chem. 2017, 15, 4595–4600. [CrossRef] 85. Makam, P.; Gazit, E. Minimalistic peptide supramolecular co-assembly: Expanding the conformational space for nanotechnology. Chem. Soc. Rev. 2018, 47, 3406–3420. [CrossRef] 86. Ikeda, M. Stimuli-responsive supramolecular systems guided by chemical reactions. Polym. J. 2019, 51, 371–380. [CrossRef] 87. Draper, E.R.; Adams, D.J. Controlling the assembly and properties of low-molecular-weight hydrogelators. Langmuir 2019, 35, 6506–6521. [CrossRef] 88. Sugiura, T.; Kanada, T.; Mori, D.; Sakai, H.; Shibata, A.; Kitamura, Y.; Ikeda, M. Chemical stimulus-responsive supramolecular hydrogel formation and shrinkage of a hydrazone-containing short peptide derivative. Soft Matter 2020, 16, 899–906. [CrossRef] 89. Ohtomi, T.; Higashi, S.L.; Mori, D.; Shibata, A.; Kitamura, Y.; Ikeda, M. Effect of side chain phenyl group on the self-assembled morphology of dipeptide hydrazides. Pept. Sci. 2020, e24200. [CrossRef] 90. Lampel, A. Biology-inspired supramolecular peptide systems. Chem 2020, 6, 1222–1236. [CrossRef] 91. Gour, N.; Kedracki, D.; Safir, I.; Ngo, K.X.; Vebert-Nardin, C. Self-assembling DNA–peptide hybrids: Morphological consequences of oligonucleotide grafting to a pathogenic amyloid fibrils forming dipeptide. Chem. Commun. 2012, 48, 5440–5442. [CrossRef][PubMed] 92. Gour, N.; Abraham, J.N.; Chami, M.; Castillo, A.; Verma, S.; Vebert-Nardin, C. Label-free, optical sensing of the supramolecular assembly into fibrils of a ditryptophan–DNA hybrid. Chem. Commun. 2014, 50, 6863–6865. [CrossRef][PubMed] 93. Nakamura, Y.; Yamada, S.; Nishikawa, S.; Matsuura, K. DNA-modified artificial viral capsids self-assembled from DNA-conjugated β-annulus peptide. J. Pept. Sci. 2017, 23, 636–643. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 9458 23 of 25

94. Matsuura, K.; Watanabe, K.; Matsuzaki, T.; Sakurai, K.; Kimizuka, N. Self-assembled synthetic viral capsids from a 24-mer viral peptide fragment. Angew. Chem. Int. Ed. 2010, 49, 9662–9665. [CrossRef][PubMed] 95. Nakamura, Y.; Inaba, H.; Matsuura, K. Construction of artificial viral capsids encapsulating short DNAs via disulfide bonds and controlled release of DNAs by reduction. Chem. Lett. 2019, 48, 544–546. [CrossRef] 96. Matsuura, K.; Watanabe, K.; Matsushita, Y.; Kimizuka, N. Guest-binding behavior of peptide nanocapsules self-assembled from viral peptide fragments. Polym. J. 2013, 45, 529–534. [CrossRef] 97. Matsuura, K.; Ueno, G.; Fujita, S. Self-assembled artificial viral capsid decorated with gold nanoparticles. Polym. J. 2015, 47, 146–151. [CrossRef] 98. Fujita, S.; Matsuura, K. Encapsulation of CdTe quantum dots into synthetic viral capsids. Chem. Lett. 2016, 45, 922–924. [CrossRef] 99. Matsuura, K.; Nakamura, T.; Watanabe, K.; Noguchi, T.; Minamihata, K.; Kamiya, N.; Kimizuka, N. Self- assembly of Ni-NTA-modified β-annulus peptides into artificial viral capsids and encapsulation of His-tagged proteins. Org. Biomol. Chem. 2016, 14, 7869–7874. [CrossRef] 100. Matsuura, K. Synthetic approaches to construct viral capsid-like spherical nanomaterials. Chem. Commun. 2018, 54, 8944–8959. [CrossRef] 101. Inaba, H.; Matsuura, K. Peptide nanomaterials designed from natural supramolecular systems. Chem. Rec. 2019, 19, 843–858. [CrossRef][PubMed] 102. Matsuura, K.; Ota, J.; Fujita, S.; Shiomi, Y.; Inaba, H. Construction of ribonuclease-decorated artificial virus-like capsid by peptide self-assembly. J. Org. Chem. 2020, 85, 1668–1673. [CrossRef][PubMed] 103. Matsuura, K. Dressing up artificial viral capsids self-assembled from C-terminal-modified β-annulus peptides. Polym. J. 2020, 52, 1035–1041. [CrossRef] 104. Kye, M.; Lim, Y.-B. Reciprocal self-assembly of peptide-DNA conjugates into a programmable sub-10-nm supramolecular deoxyribonucleoprotein. Angew. Chem. Int. Ed. 2016, 55, 12003–12007. [CrossRef] 105. Kye, M.; Lim, Y. Synthesis and purification of self-assembling peptide-oligonucleotide conjugates by solid-phase peptide fragment condensation. J. Pept. Sci. 2018, 24, e3092. [CrossRef] 106. Basavalingappa, V.; Bera, S.; Xue, B.; Azuri, I.; Tang, Y.; Tao, K.; Shimon, L.J.W.; Sawaya, M.R.; Kolusheva, S.; Eisenberg, D.S.; et al. Mechanically rigid supramolecular assemblies formed from an Fmoc-guanine conjugated peptide nucleic acid. Nat. Commun. 2019, 10, 5256. [CrossRef] 107. Basavalingappa, V.; Guterman, T.; Tang, Y.; Nir, S.; Lei, J.; Chakraborty, P.; Schnaider, L.; Reches, M.; Wei, G.; Gazit, E. Expanding the functional scope of the Fmoc-diphenylalanine hydrogelator by introducing a rigidifying and chemically active urea backbone modification. Adv. Sci. 2019, 6.[CrossRef] 108. Arakawa, H.; Takeda, K.; Higashi, S.L.; Shibata, A.; Kitamura, Y.; Ikeda, M. Self-assembly and hydrogel formation ability of Fmoc-dipeptides comprising α-methyl-L-phenylalanine. Polym. J. 2020, 52, 923–930. [CrossRef] 109. Daly, M.L.; Gao, Y.; Freeman, R. Encoding reversible hierarchical structures with supramolecular peptide– DNA materials. Bioconjug. Chem. 2019, 30, 1864–1869. [CrossRef] 110. Freeman, R.; Han, M.; Álvarez, Z.; Lewis, J.A.; Wester, J.R.; Stephanopoulos, N.; McClendon, M.T.; Lynsky, C.; Godbe, J.M.; Sangji, H.; et al. Reversible self-assembly of superstructured networks. Science 2018, 362, 808–813. [CrossRef] 111. Rha, A.K.; Das, D.; Taran, O.; Ke, Y.; Mehta, A.K.; Lynn, D.G. Electrostatic complementarity drives amyloid/nucleic acid co-assembly. Angew. Chem. Int. Ed. 2020, 59, 358–363. [CrossRef][PubMed] 112. Mou, Y.; Yu, J.-Y.; Wannier, T.M.; Guo, C.-L.; Mayo, S.L. Computational design of co-assembling protein–DNA nanowires. Nature 2015, 525, 230–233. [CrossRef][PubMed] 113. Clarke, N.D.; Kissinger, C.R.; Desjarlais, J.; Gilliland, G.L.; Pabo, C.O. Structural studies of the engrailed homeodomain. Protein Sci. 1994, 3, 1779–1787. [CrossRef][PubMed] 114. Dirks, R.M.; Pierce, N.A. Triggered amplification by hybridization chain reaction. Proc. Natl. Acad. Sci. USA 2004, 101, 15275–15278. [CrossRef] 115. Fu, T.; Lyu, Y.; Liu, H.; Peng, R.; Zhang, X.; Ye, M.; Tan, W. DNA-based dynamic reaction networks. Trends Biochem. 2018, 43, 547–560. [CrossRef] 116. McMillan, J.R.; Hayes, O.G.; Remis, J.P.; Mirkin, C.A. Programming protein polymerization with DNA. J. Am. Chem. Soc. 2018, 140, 15950–15956. [CrossRef] 117. Figg, C.A.; Winegar, P.H.; Hayes, O.G.; Mirkin, C.A. Controlling the DNA hybridization chain reaction. J. Am. Chem. Soc. 2020, 142, 8596–8601. [CrossRef] Int. J. Mol. Sci. 2020, 21, 9458 24 of 25

118. Xu, Y.; Jiang, S.; Simmons, C.R.; Narayanan, R.P.; Zhang, F.; Aziz, A.-M.; Yan, H.; Stephanopoulos, N. Tunable nanoscale cages from self-assembling DNA and protein building blocks. ACS Nano 2019, 13, 3545–3554. [CrossRef] 119. Subramanian, R.H.; Smith, S.J.; Alberstein, R.G.; Bailey, J.B.; Zhang, L.; Cardone, G.; Suominen, L.; Chami, M.; Stahlberg, H.; Baker, T.S.; et al. Self-assembly of a designed nucleoprotein architecture through multimodal interactions. ACS Cent. Sci. 2018, 4, 1578–1586. [CrossRef] 120. Brodin, J.D.; Ambroggio, X.I.; Tang, C.; Parent, K.N.; Baker, T.S.; Tezcan, F.A. Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nat. Chem. 2012, 4, 375–382. [CrossRef] 121. Brodin, J.D.; Carr, J.R.; Sontz, P.A.; Tezcan, F.A. Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties. Proc. Natl. Acad. Sci. USA 2014, 111, 2897–2902. [CrossRef][PubMed] 122. Brodin, J.D.; Smith, S.J.; Carr, J.R.; Tezcan, F.A. Designed, helical protein nanotubes with variable diameters from a single building block. J. Am. Chem. Soc. 2015, 137, 10468–10471. [CrossRef][PubMed] 123. Keller, A.; Linko, V. Challenges and perspectives of DNA nanostructures in biomedicine. Angew. Chem. Int. Ed. 2020, 59, 15818–15833. [CrossRef][PubMed] 124. Ishihara, K.; Ohara, S.; Yamamoto, H. 3,4,5-trifluorobenzeneboronic acid as an extremely active amidation catalyst. J. Org. Chem. 1996, 61, 4196–4197. [CrossRef] 125. Pattabiraman, V.R.; Bode, J.W. Rethinking amide bond synthesis. Nature 2011, 480, 471–479. [CrossRef]

126. Opie, C.R.; Noda, H.; Shibasaki, M.; Kumagai, N. All non-carbon B3NO2 exotic heterocycles: Synthesis, dynamics, and catalysis. Chem. Eur. J. 2019, 25, 4648–4653. [CrossRef] 127. Liu, Z.; Noda, H.; Shibasaki, M.; Kumagai, N. Catalytic oligopeptide synthesis. Org. Lett. 2018, 20, 612–615. [CrossRef] 128. Todorovic, M.; Perrin, D.M. Recent developments in catalytic amide bond formation. Pept. Sci. 2020, 112. [CrossRef] 129. Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell, 4th ed.; Garland Science: New York, NY, USA, 2002; ISBN 978-0-8153-3218-3. 130. Suhanovsky, M.M.; Teschke, C.M. Nature’s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold. Virology 2015, 479–480, 487–497. [CrossRef] 131. Wu, H.; Fuxreiter, M. The structure and dynamics of higher-order assemblies: Amyloids, signalosomes, and granules. Cell 2016, 165, 1055–1066. [CrossRef] 132. Hoffman, D.P.; Shtengel, G.; Xu, C.S.; Campbell, K.R.; Freeman, M.; Wang, L.; Milkie, D.E.; Pasolli, H.A.; Iyer, N.; Bogovic, J.A.; et al. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells. Science 2020, 367.[CrossRef][PubMed] 133. Shin, Y.; Brangwynne, C.P. Liquid phase condensation in cell physiology and disease. Science 2017, 357. [CrossRef][PubMed] 134. Kroschwald, S.; Alberti, S. Gel or die: Phase separation as a survival strategy. Cell 2017, 168, 947–948. [CrossRef][PubMed] 135. Yoshizawa, T.; Nozawa, R.-S.; Jia, T.Z.; Saio, T.; Mori, E. Biological phase separation: Cell biology meets biophysics. Biophys. Rev. 2020, 12, 519–539. [CrossRef] 136. Trivedi, P.; Palomba, F.; Niedzialkowska, E.; Digman, M.A.; Gratton, E.; Stukenberg, P.T. The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nat. Cell Biol. 2019, 21, 1127–1137. [CrossRef] 137. Herzel, L.; Ottoz, D.S.M.; Alpert, T.; Neugebauer, K.M. Splicing and transcription touch base: Co- transcriptional spliceosome assembly and function. Nat. Rev. Mol. Cell Biol. 2017, 18, 637–650. [CrossRef] 138. Sanulli, S.; Trnka, M.J.; Dharmarajan, V.; Tibble, R.W.; Pascal, B.D.; Burlingame, A.L.; Griffin, P.R.; Gross, J.D.; Narlikar, G.J. HP1 reshapes nucleosome core to promote phase separation of heterochromatin. Nature 2019, 575, 390–394. [CrossRef] 139. Guo, Y.E.; Manteiga, J.C.; Henninger, J.E.; Sabari, B.R.; Dall’Agnese, A.; Hannett, N.M.; Spille, J.-H.; Afeyan, L.K.; Zamudio, A.V.; Shrinivas, K.; et al. Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 2019, 572, 543–548. [CrossRef] 140. Du, Y.; Dong, S. Nucleic acid biosensors: Recent advances and perspectives. Anal. Chem. 2017, 89, 189–215. [CrossRef] Int. J. Mol. Sci. 2020, 21, 9458 25 of 25

141. Rubert Pérez, C.M.; Stephanopoulos, N.; Sur, S.; Lee, S.S.; Newcomb, C.; Stupp, S.I. The powerful functions of peptide-based bioactive matrices for regenerative medicine. Ann. Biomed. Eng. 2015, 43, 501–514. [CrossRef] 142. Gelain, F.; Luo, Z.; Zhang, S. Self-assembling peptide EAK16 and RADA16 nanofiber scaffold hydrogel. Chem. Rev. 2020.[CrossRef][PubMed] 143. Armstrong, J.P.K.; Keane, T.J.; Roques, A.C.; Patrick, P.S.; Mooney, C.M.; Kuan, W.-L.; Pisupati, V.; Oreffo, R.O.C.; Stuckey, D.J.; Watt, F.M.; et al. A blueprint for translational regenerative medicine. Sci. Transl. Med. 2020, 12, eaaz2253. [CrossRef][PubMed] 144. Cutler, J.I.; Auyeung, E.; Mirkin, C.A. Spherical nucleic acids. J. Am. Chem. Soc. 2012, 134, 1376–1391. [CrossRef][PubMed] 145. Tan, X.; Jia, F.; Wang, P.; Zhang, K. Nucleic acid-based drug delivery strategies. J. Control. Release 2020, 323, 240–252. [CrossRef] 146. Jiang, Z.; Thayumanavan, S. Noncationic material design for nucleic acid delivery. Adv. Therap. 2020, 3, 1900206. [CrossRef] 147. Roberts, T.C.; Langer, R.; Wood, M.J.A. Advances in oligonucleotide drug delivery. Nat. Rev. Drug Discov. 2020, 19, 673–694. [CrossRef]

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