DNA Origami Meets Polymers: A Powerful Tool for the Design of Defined Nanostructures

Abstract The combination of DNA origami nanostructures and polymers provides a new possibility to access defined structures in the 100 nm range. In general, DNA origami serves as a versatile template for the highly specific arrangement of polymer chains. Polymer‐DNA hybrid nanostructures can either be created by growing the polymer from the DNA template or by attaching preformed polymers to the DNA scaffold. These conjugations can be of a covalent nature or be based on base‐pair hybridization between respectively modified polymers and DNA origami. Furthermore, the negatively charged DNA backbone permits interaction with positively charged polyelectrolytes to form stable complexes. The combination of polymers with tuneable characteristics and DNA origami allows the creation of a new class of hybrid materials, which could offer exciting applications for controlled energy transfer, nanoscale organic circuits, or the templated synthesis of nanopatterned polymeric structures.


Introduction
Thef abrication of functional nanoparticles and defined nanoscale surfaces represents an intensively investigated topic of current research. Besides the synthesis of such materials,t he improvement in the fabrication of smaller and more precise geometries as well as the development of precisely addressable surfaces is also of interest. Significant improvements in such fabrication techniques could be of further usage for, for example,r educing the size of data storage,o ptical devices,o rt he development of new drugdelivery systems. [1][2][3] Such nanostructures can be fabricated in many ways,b ut two of the most important methods are lithography and selfassembly.L ithography,a sat op-down technique,e nables manipulation of larger objects to result in smaller-size geometries with the desired shape. [4] Nevertheless,i to ften requires expensive and complicated setups,t hus making the fabricated samples expensive and not suitable for the largescale fabrication of nanostructures. [5] In contrast, self-assembly,a sab ottom-up process,r elies on the interactions of the assembling units without any external stimuli, which will be discussed in the following only for small molecules. [4] Such moieties can be,f or example,b ased on hydrogen bonding, [6] van der Waals forces, [7] hydrophobic and hydrophilic [8][9] interactions,o rp-p stacking. [10] During the self-assembly processes of synthetic molecules,v arious desired structures could be formed, which makes this process alow cost and fast alternative compared to lithography. [11] However,n ot all geometries can be realized in this way.
One prominent example for av ersatile self-assembling process in nature is the formation of the DNAd ouble helix, which is based on hydrogen bonding between complementary base pairs.I n1 982, Seeman took inspiration from such processes and realized the folding of DNAi nto designed superstructures. [12,13] This idea was further expanded by Rothemund in 2006, by establishing the so-called DNA origami technology,w hich led to ab reakthrough in the construction of DNAobjects. [14] In this approach, al ong, circular singlestranded DNA( "scaffold DNA" )i s folded into ad istinct shape with the help of as et of short "staple strands". These staple strands are designed to hybridize to complementary sequences within the scaffold DNA. Elongating particular staple strands by short oligonucleotides results in surfaceprotruding single-stranded DNA (ssDNA), which can subsequently undergo hybridization to additional molecules. ( Figure 1) Thus,D NA origami provides ap recisely addressable surface and has been shown to be apowerful tool for the distinct positioning of, for example,n anoparticles in ap redefined manner. [12,15] In this Minireview,wefocus on the functionalization of DNAo rigami nanostructures with synthetic polymers or polymer-oligonucleotide conjugates to afford unique hybrid nanostructures that are very challenging to achieve with other techniques. However,i nc ontrast to previous contributions,t he pure self-assembly behavior of polymer-DNAc onjugates will not be discussed. [16][17][18] However,arange of methods for the attachment of polymers onto DNAn anostructures in a predesigned manner is described in detail. Additionally, the advancements as well as the limitations of the functionalization of DNAo rigami is discussed and compared to lithography and traditional self-assembly methods.Finally,we show that the functionalization of DNAo rigami can be ap owerful tool for the preparation of polymeric nanostructured objects.
The combination of DNAorigami nanostructures and polymers provides anew possibility to access defined structures in the 100 nm range.Ing eneral, DNAo rigami serves as aversatile template for the highly specific arrangement of polymer chains.P olymer-DNAhybrid nanostructures can either be created by growing the polymer from the DNAt emplate or by attaching preformed polymers to the DNA scaffold. These conjugations can be of acovalent nature or be based on base-pair hybridization between respectively modified polymers and DNAo rigami. Furthermore,t he negatively charged DNAb ackbone permits interaction with positively charged polyelectrolytes to form stable complexes.T he combination of polymers with tuneable characteristics and DNAo rigami allows the creation of anew class of hybrid materials,whichcould offer exciting applications for controlled energy transfer,n anoscale organic circuits,ort he templated synthesis of nanopatterned polymeric structures.

Polymers and DNA Origami:How to Bridge the Fields
Thec ombination of polymers and DNAo rigami has the power to merge the fields of synthetic and natural macromolecules,w hile getting the best of both worlds. [19,20] On the one hand, the unprecedent addressability of DNAo rigami may organize polymers on the nanoscale into structures as polymers are typically known for entanglement and, thereby, pave the way for nanotechnological devices and structurefunction investigations.O nt he other hand, there is al arge pool of polymers with av ast range of appealing and adjustable characteristics such as various charges,hydrophobicity or hydrophilicity,a sw ell as stimulus-responsiveness,a nd they may also stabilize DNAobjects.T here is aversatile range of ways to guide the process and achieve this fusion ( Figure 2). Tw of undamental strategies have to be distinguished here: Either the polymer is grown in situ on the DNAo rigami template (see Section 2.1) or the polymer is preformed and modified prior to conjugation to the DNAp latform (see Section 2.2).
On the molecular level, the underlying principles for polymer attachment are manifold. Thep olymer can be electrostatically trapped to the negatively charged DNA backbone by the incorporation of respective positive counter charges or be bound to the DNAo rigami surface through base-pair hybridization. Theo ligonucleotides required for this can be introduced by click reactions,b ye stablished bioconjugation techniques,o rg rown from nucleotides.F urthermore,h ydrophobic interactions between the implemented polymers can be exploited to arrange polymer-DNA constructs into higher ordered structures (see Section 2.3). However,the rather small number of publications in the field of DNAorigami and polymer hybrids gives afirst hint of how challenging this topic seems to be.N ot only does synthesis suffer from various issues,s uch as solubility issues or steric Nadine Hannewalds tudied chemistry at the Friedrich Schiller University in Jena and graduated in 2018 in the field of organic and macromolecular Chemistry.S he is currently working as aP hD student in the group of Prof. Schubert, where she focuses on the synthesis of well-defined polymer architecturesf or attachment to DNA origami.

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Minireviews 6220 www.angewandte.org hindrance of both the DNAa nd polymer reactive sites,b ut the characterization techniques are also very limited. One of the key drawbacks is the typically extremely low amount of DNAo rigami structures available,w hich impedes or even prohibits,f or example,f reeze-pump-thaw cycles for controlled radical polymerizations,s ufficient amounts of attached initiator sites,o rc onventional polymer analysis by size-exclusion chromatography (SEC), nuclear magnetic resonance (NMR), or dynamic light scattering (DLS).

Polymer Growth from DNA Origami
Theg rafting from strategy is ac onvenient approach towards the synthesis of biomolecule-polymer constructs with tailored properties,w hich are characterized by facile purification of the conjugate and commonly ahigh graft density. [21] Controlled polymerization techniques have emerged as ap owerful method to create polymers of controlled molecular weights and well-defined architectures. [22] Among others, atom transfer radical polymerization (ATRP) provides the possibility to conduct the polymerization under biologically relevant conditions that are suited to the stability of biomolecules,alow concentration of functional groups,o r the presence of salts when working with buffers. [23] However, successful polymerization from the biomolecule surface demands the installation of reactive handles which serve as initiator sites.W ee mployed the highly precise scaffold of DNAo rigami to anchor ATRP initiators at predefined positions and, thereby,a chieve directed polymer growth on the nanoscale (Figure 3). [24] DNAo rigami sheets were equipped with different patterns of surface-protruding,s hort oligonucleotide sequences.C omplementary oligonucleotides were modified with ATRP initiators and attached to the DNA origami template by base-pair hybridization. This macroinitiator was then utilized to induce the polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGME-MA). This monomer was chosen because of its biocompatibility as well as its solubility in water, and the rather bulky side chains were considered to facilitate monitoring of the polymerization process by atomic force microscopy (AFM). Furthermore,t he presence of sacrificial initiators (excess amount of free initiator DNAn ot attached to the DNA origami)w as found to be crucial for successful polymer growth. Visualization of the origami structures by AFM, in particular recording the height profile,r evealed the appearance of new objects where initiator sites were located at defined positions on the DNAn anotile.F urthermore,t hese objects have different mechanical properties which correspond to features of soft polymeric materials,s uch as PEGMEMA. Nevertheless,t ypical characterization of the polymer,s uch as determination of the chain length or dispersity by size-exclusion chromatography,i sn ot feasible here because of very low quantities.T he incorporation of the bifunctional monomer PEG dimethacrylate (PEGDMA) to the polymerization process led to ac ross-linked polymer, whose structure could be preserved even after removal of the DNAt emplate.
An essentially different polymerization technique,b ut also ag rafting-from strategy from DNAn anostructures,w as introduced by Ding and co-workers. [25] They decorated adouble-stranded DNAtemplate with guanine-rich oligonucleotide sequences,t he so-called DNAzymes,w hich are capable of mimicking the activity of the enzyme horseradish  Reprinted from Ref. [24] with permission.

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Chemie peroxidase (HRP). Upon incorporation of the cofactor hemin and addition of hydrogen peroxide,t he active DNAzyme catalyzes the polymerization of aniline.T hus,1Dpolyaniline (PANI) structures were formed by a para-coupling reaction, wherein the generated aniline radicals diffuse to the charged DNAsurface.The regioselective formation of PA NI was then transferred to 2D origami triangles ( Figure 4). [26] However, the use of DNAo rigami structures was challenging and required optimization of the reaction conditions:W hereas ahigh ionic strength disfavored the para-coupling reaction of PA NI, an insufficient Mg 2+ concentration compromises the stability of the DNAf olding.B yA FM imaging, the group could show that polymer growth was favored around the DNAzymes and did not grow over the DNAzyme-free regions.T hus,s tructural information transfer from the origami pattern to PA NI was achieved, thereby leading to ap olymer of predesigned geometry.F urthermore,t he reversible redox behavior of polyaniline,which can be triggered by pH changes,renders these conductive hybrid objects promising candidates for the fields of electronics,sensors,and energy storage.
Therelatively simple and tolerant polymerization strategy was also applied to the polymerization of dopamine on DNA origami nanostructures. [27] Polydopamine is amussel-inspired polymer which has aroused great interest among material scientists because of its excellent capability for surface functionalization. [28,29] However,t he self-polymerization of dopamine and the not yet fully elucidated multifaceted polymer structure hamper its full potential. By employing the same DNAzymes as described above,wecould induce and promote polydopamine formation on a2 DD NA nanosheet. It was essential to conduct the polymerization in an acidic milieu to suppress the self-polymerization of dopamine and to gain control over the process.Different polydopamine shapes and sizes were obtained by arranging the catalytic centers in different patterns on the origami scaffold, and the reaction kinetics could be manipulated by altering the ionic strength and hydrogen peroxide concentration. Thef abricated polydopamine nanostructures could serve as a" supramolecular glue", thus guiding the origami conformation. This is an illustrative example of how the DNAtemplate can affect the polymer formation and vice versa. In af ollow-up study,3 D origami structures were decorated with ap hotosensitizer, which was trapped at distinct positions by guanine-rich oligonucleotides (G-quadruplexes;F igure 5). [30] Upon irradiation with visible light, dopamine was oxidized and polydopamine was deposited around the reaction centers.A saconsequence of the light stimulus,t he presence of hydrogen peroxide is no longer needed, which keeps the system simple. In addition, the polymerization process could be temporally controlled by simply switching the light on and off.Inthis way, photopatterned 3D nanostructures with dimensions far below 100 nm were created, which could not only preserve the DNA template in salt-depleted media but they could also be released from the template under strong acidic conditions.

Polymer Attachment to DNA Origami
In all the examples discussed above,t he polymer chain was grown from the DNAorigami surface in distinct patterns, either covalently attached to the initiators by ac ontrolled polymerization technique or noncovalently deposited next to the initiators on the DNAt emplate.I nc ontrast to this methodology,one can also make use of apreformed polymer, artificially synthesized or biologically derived, with graftsuitable reactive handles and attach it to DNAn anostructures.Besides the rather intuitive idea of trapping apositively charged polymer by electrostatic interactions,i ti sa lso appealing to hybridize polymers by base-pair recognition or to exploit the attractive area of click chemistry.H owever, many of the studies illustrate the boundaries of the bespoke strategies,s uch as the steric hindrance of polymers,t heir  DNA origami by DNAzymes to create highly precise hybrid objects. [27] B) Photoinduced formation of polydopamine on 3D DNA origami under spatiotemporal control with the help of al ocally trapped photosensitizer.Reprinted from Ref. [30] with permission.

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Minireviews 6222 www.angewandte.org solubility,a nd the stability of DNA, which all impair successful conjugation.

Electrostatic Interactions
Thei onic nature of the phosphate backbone of DNA makes it possible to attach polymers through electrostatic interactions to DNAo rigami. Usually,t he DNAo rigami structures are stabilized by the divalent cation Mg 2+ ,w hich screens the negatively charged phosphate backbone of the DNAs equence to compensate charge repulsion between closely packed DNAs trands.I namultitude of studies,t he applied polymers comprise amino moieties in the side chain or backbone that undergo quaternization of the nitrogen atom when applied in acidic media. These polycations can then interact with DNAo rigami through ionic interactions.
Based on this idea, Kiviaho et al. investigated the electrostatic binding between a6 0-helix-bundled DNAn anostructure and cationic block copolymers. [31] To assess the effect of the polymer structure on the binding affinity,t he authors synthesized AB-and ABA-type copolymers by ATRP.F or this,t hey utilized ar espective mono-and bifunctional PEGbased macroinitiator to polymerize 2-dimethylaminoethyl methacrylate (PDMAEMA), where the PEG moiety was intended to increase the poor biocompatibility of PDMAE-MA. Coating was achieved by simply mixing the compounds under mild acidic conditions to ensure protonation of the amines.I tc ould be demonstrated that all the polymers had as uitable binding efficiencyb ut, interestingly,t he block structure only had am inor impact. Instead, the ratio of total number of polymer amines and the total number of phosphates in DNA( referred to as the N/P ratio) was pivotal, irrespective of the arrangement of the nitrogen atoms in the polymer.M oreover,v arious polymer coatings were suited to control the activity of enzyme-loaded DNAo rigami nanocontainers,a si ndicated by the bioluminescence reaction of luciferase enzymes.Inafurther study,commercially available linear polyethyleneimine (LPEI) and chitosan as an atural polymer were applied to form polyplexes with DNAorigami nanostructures ( Figure 6). [32] Theauthors aimed to investigate several factors that might have an impact on the origami stability under physiological conditions,s uch as degree of polymerization, charge density,a nd nitrogen to phosphate ratio.T hree different DNAo bjects were synthesized and applied for this purpose:an anorod, an anobottle,a nd aw ireframe origami structure.A fter simple mixing of the DNAa nd polymer compounds,s uccessful coating was demonstrated with the PicoGreen assay,w hich relies on the intercalation of the dye into the DNAd ouble helix while exhibiting strong fluorescence.A saconsequence of the polycation coating, PicoGreen was expelled from the polymer-DNAc omplex, thereby resulting in ad ecrease in the fluorescence.A lthough bare origami could be imaged by negative-stain transmission electron microscopy (nsTEM), the staining of the LPEI-modified origami was only possible after removing the polymer coating by treatment with polyanionic dextran sulfate;t his revealed intact origami structures and, thus,i ndirectly indicated successful encapsulation by the polymer.I tc ould be shown that LPEI protects the structural integrity of the DNAo rigami more efficiently than chitosan and that this ability strongly depends on the nitrogen to phosphate (N/P) ratio.H owever,i tm ust be considered that the unique addressability of the DNAorigami surface might be masked by the polymer coating.
In 2017, two studies investigated the use of PEGoligolysine-based copolymers to protect DNAo rigami structures against low-salt denaturation and nuclease degradation, while the lysine block provides the positive charges to electrostatically interact with the DNAo bject, and the PEG is envisioned to have as hielding effect. [33,34] TheS chmidt group synthesized poly(ethylene glycol)-b-poly(l-lysine) (PEG 12kDa -PLys 18 )b yr ing-opening polymerization of N etrifluoroacetyl-l-lysine N-carboxyanhydride initiated by an amine-terminated 12 kDa PEG macroinitiator.Incontrast to bare origami structures,p olymer-coated objects resisted the treatment of DNase I, fetal bovine serum (FBS), and low salt levels,a nd maintained structural integrity.H owever,t he attachment of sterically demanding gold nanoparticles (AuNP) did not survive the process of polyplex formation; detachment could by visualized by transmission scanning electron microscopy (tSEM). Thep roblem could be circumvented by employing shorter PEG chains,which still offer the same protection efficiency.T hese findings are in agreement with as imilar study by Shih and co-workers,w ho examined the beneficial contribution of the PEG 5kDa PLys 10 polymer coating to the origami stability.They could further prove that surface addressability of the DNAn anostructures was not constrained by the polymer film and immobilized ligands were capable of targeting receptors,t hereby leading to cellular uptake of the hybrid objects.V ery recently,G ang and co-workers endeavored to also push the limits of the stability of DNAa ssembly in complex biological fluids (Figure 7). [35] They put anovel class of polycationic polymers, namely peptoids,t ot he test. Peptoids are emerging peptidomimetics,whose side chains are not appended to the a-carbon but to the nitrogen atom of the peptide backbone,t hus, preventing secondary structure formation through hydrogen bonding and providing proteolysis resistance.Inline with the approaches discussed above,the group explored the effect of peptoid architecture and sequence dependencyo nt he origami stability.F or this,t hey synthesized, by solid-phase peptoid synthesis,b rush-and block-like peptoids that were built from positively charged monomers (electrostatic DNA complexation) and neutral oligo(ethyleneoxy) moieties (surface passivation). They could demonstrate that brush-like peptoids were superior in protecting wireframed octahedrashaped DNAo rigami. Moreover,t he capability of these structures to serve as adrug carrier with controlled release of doxorubicin was shown, which had not been achieved before. All these coating strategies are rather easily achieved by simply mixing the origami nanostructures with an excess of polymer,but they lack the possibility to arrange the polymer in distinct patterns.

Oligonucleotide Hybridization
Thea ttachment of polymers through ionic interactions, which are often just used to stabilize DNA, is very advantageous in terms of synthesis as well as the ease and straightforward fusion of ap olymer and DNAo rigami. Nonetheless,t his strategy does not consider the unique addressability provided by the DNAo rigami scaffold;o n the contrary,i tm ight even hinder it. Hence,t he linkage of polymers to DNAo bjects by complementary base-pair recognition allows the highly precise positioning of single polymer chains and overcomes their lack of intrinsic selfassembly properties.T oe quip polymers with the necessary handles,namely,oligonucleotides that are complementary to ssDNAs equences on the origami surface,o ne can either functionalize the polymerse nd group or the side chains accordingly.I th as been proven useful to either "click" the oligonucleotide to the polymer or to grow oligonucleotides directly from the polymer backbone.I nb oth cases,h ybridizing the respective polymer to the DNAo rigami is always reversible and should permit programmed switching.
Gothelf and co-workers see agreat prospective in binding conjugated and, therefore,p otentially conducting polymers on DNAo rigami templates to build molecular-scale elec-tronic or optical wires. [36] Fort his purpose,t hey synthesized ac onjugated poly(phenylenevinylene) polymer with alkoxy side chains (APPV) from adithiocarbamate precursor.E ach phenylene unit in the backbone bears atriethylene glycol side chain and with the help of protective group chemistry,asmall number of hydroxy groups were employed to attach the polymer to the solid support;t he remaining hydroxy groups were used in automated solid-phase DNAsynthesis to graft 9mer oligonucleotides.Bythis approach, they obtained afully water-soluble brush polymer with ssDNAextending from the majority of the repeating units.However,the size distribution was rather broad, as characterized by gel-permeation chromatography (GPC;3 40-3300 kDa) and AFM (lengths in the range of 20 nm to 200 nm), which the authors explain through partial degradation during purification. By equipping 2D and 3D DNAo rigami templates with complementary oligonucleotide sequences,t hey could link single polymer chains to the template in different geometries.M oreover,t hey could observe Fçrster resonance energy transfer (FRET) between the attached polymer (donor) and aco-immobilized acceptor dye,t hereby proving that absorption and emission of the polymer backbone is not harmed by the applied methods. Further studies that exploit this strategy towards the development of nanocircuits are discussed in Section 3.
In contrast to the rather sophisticated and challenging solid-phase synthesis of oligonucleotides directly from the polymer backbone,o ne can also furnish the polymer with as uitable end group and "click" it to the respective ssDNA. In this context, copper-catalyzed azide-alkyne reactions (CuAAC) [37] as well as ac opper-free variant involving as train-promoted azide-alkyne click reaction (spAAC) [38,39] have been utilized in different studies.For the development of aDNA origami assisted electrooptical modulator, Canary and co-workers equipped two different kinds of organic semiconductors,n amely oligomers of poly(phenylene vinylene) (HPV) and oligoaniline (OANI), with ssDNAstrands,which allows their attachment to aD NA origami scaffold (Figure 8). [37] By this approach, the symmetric oligomers with azide groups at each end were "double-clicked" by CuAAc to oligonucleotide strands containing ap ropargyl residue.C onsequently,the obtained structure consisted of apolymer with oligonucleotide sequences at both ends.B yh ybridizing the polymer-DNAc onstructs to aDNA origami frame with four complementary anchor strands,t he semiconductors were brought into proximity,thereby forming across-like structure. By tuning the oxidation state of polyaniline,t he energy transfer from HPV to OANI could be tuned, as visualized by an altered fluorescence signal. However,t he hybridization efficiency of only 20 %c orrectly formed polymer-DNA origami structures (determined by AFM) illustrates how challenging the formation of these hybrid objects is,although base-pair hybridization is often assumed to be straightforward. Mertig and co-workers also employed click reactions to conjugate conducting polymers in distinct patterns to an origami surface (Figure 9). [38] Fort his,t hey synthesized welldefined thiophene-based polymers with dispersities between 1.1 to 1.3 by Kumada polycondensation. Oligoethylene glycol bearing side chains ensured water solubility of the polymer Figure 7. "Brush-" and "block-type" peptoids should lead to different surface coatings on octahedra-shaped DNA origami. Reprinted from Ref. [35] with permission.

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Chemie and thereby allowed reaction of the azide-functionalized polymer and dibenzocyclooctyne-end-capped oligonucleotide in aqueous solution. It is noteworthy that the degree of functionalization of the polymers is only in the moderate range of 38-71 %. However, unfunctionalized polymer chains are not considered to participate in, or even harm, further transformations.T hree different oligonucleotide sequences were conjugated to the polymers and were attached to three different DNAo rigami pads with patterns of respective handles to study sequence-hybridization effects.V irtually all the pads displayed at least one attached object, but the overall occupation probability per handle was roughly one third. For example,4out of 14 handles on one origami pad displayed an attached object. This again indicates that hybridization of polymers to DNAsurfaces is difficult and sterically demanding. Applying surfactants to polymer-decorated origami was accompanied by ab lue-shifted increase in the fluorescence and, thus,i ndirectly showed that interchain p-p stacking of polythiophene units occurs.T his feature might offer the possibility to fine-tune optical properties on amolecular level.

Higher Order Structures
In addition to the attachment methods discussed above, one can also make use of the hydrophobic effect to form higher ordered structures built from DNAo rigami and polymers.B yapplying hydrophobic polymers to DNAs caffolds,surface properties can be altered significantly and, thus, self-assembly of amphiphilic structures can be induced.
In 2015, Liu and co-workers showed that attaching hydrophobic dendrons to DNAorigami rectangles could lead to the formation of surface areas with ah igh local concentration of hydrophobic molecules,w hich, as ar esult of the hydrophobic effect, guided origami folding into various thermodynamically stable products.P oly(aryl ether) dendrons were conjugated to oligonucleotides through solidphase synthesis,w hereas modification with oligo(ethylene glycol) tails should increase the water solubility of the dendrons.Upon traditional origami annealing in the presence of both elongated capture strands (handles on the origami) and oligonucleotide-bearing dendrons (complementary to handles on the origami), sandwich-like structures were created. In afollow-up study,the same group created polymer vesicles on the shell of aD NA origami cube ( Figure 10). [40] Thea ttachment of the above-mentioned hydrophobic dendrons to origami cubes led to aggregation and precipitation events,m ost likely because of p-p stacking between several cuboid frames (frame-frame interactions). Thea ddition of asecond hydrophobic dendron, the so-called principal amphiphile (PA), to the amphiphilic construct breaks the frame-frame interactions and promotes stronger PA -frame interactions,t hereby resulting in the formation of heterovesicles.T od emonstrate the applicability of this process to different molecules,t he dendrons were substituted by polymers:D NA cuboids were covered with thermoresponsive poly(propylene oxide) (PPO) and upon heating, the polymer became hydrophobic and, thus,guided asecond PPO polymer to form heterovesicles. Reprinted from Ref. [37] with permission.C opyright (2020) American Chemical Society. Figure 9. A) spAAC reaction with acyclooctyne-functionalized oligonucleotide and azide-functionalized polythiophene. B) The three different DNA origami pad types. C) Illustration of aggregated P3(EO) 3 Tonthe DNA origami (left) and the deaggregated structure (right) after the addition of surfactant. Printed from Ref. [38] with permission.C opyright (2020) American Chemical Society. This is an impressive example of higher order assemblies based on polymer-decorated DNAo rigami structures;h owever,the intricate technique requires astrong background in the field of frame-guided assembly to be successful. Theuse of hydrophobic interactions between polymers attached to 3D DNAn anostructures was also shown to yield larger DNA micelles. [41,42] Fort his purpose,D NA nanostructures of three different forms (trigonal prism, cube,a nd pentagonal prism) were decorated with oligonucleotides covalently linked to hexaethylene phosphate to yield DNAn anostructures with polymer strands. [41] It could be revealed that the number of hexaethylene phosphate repeating units is crucial for the micellization:A tl east six of these repeating units are required to form higher ordered structures,w ith micellar structures being observed when the number of repeating units is increased to at least eight. [41] Not only were micelles with cubic DNAs tructures synthesized, but trigonal and pentagonal prisms were also obtained. TEM, AFM and DLS were utilized to compare the micelles of the different DNA structures,w hich revealed that they appear to have approximately the same size and that the size is only influenced by the number of repeating units of the hexaethylene phosphate. Thegroup further investigated the influence of acombination of hydrophobic and hydrophilic repeating units in the DNA polymer conjugates attached to the prismatic structures. [42] Thehydrophobic block consisted of 1,12-dodecanediol (HE), and the hydrophilic block was represented by hexaethyloxy glycol (HEG). First experiments combined the cubic DNA structure with four DNAcopolymer strands,which consisted of six HE and six HEG units,i nd ifferent orders.N otably, higher mobility in the gel electrophoresis was observed as the HE block length was increased, which was explained by the folding of the polymer chains into the cage structure. [42] The nanostructure with the HE 6 HEG 6 block copolymer was exceptional, as it formed rings of three to five polymerdecorated DNAc ubes in ad oughnut-like fashion instead of the expected micellar structures.B yincreasing the length of the hydrophilic HEG block, the diameter of the ringlike assemblies could be increased, in contrast to the HE 6 HEG 6 block copolymer, which indicates that the HEG block acts as as pacer. [42] Remarkably,t he formation of micelles was not observed when (block) copolymers consisting of hydrophobic and hydrophilic units were used. These can only be observed in the case of hydrophobic polymers. [42] Thep reviously discussed examples show that not only can DNAo rigami be utilized to direct polymers into larger structures and desired shapes,b ut polymer-polymer interactions also allow macromolecular structures to be created with prior-folded DNA origami.

Next-Generation Polymeric Hybrid Materials: Fields of Application
In the previous section, we highlighted different techniques for linking polymers and DNAo rigami as well as the influence on each other. Although there are fewer examples than one might expect regarding the potential provided by these materials,a nd although there are still some challenges to overcome,s everal studies report the first steps towards future applications and prospects.
Gothelf and co-workers exploited their system of attaching aconjugated brush-like polymer with oligonucleotide side chains onto DNAo rigami tiles to contribute to the area of nanophotonic and nanoelectronic devices.T hey not only attached the polymer to the origami platform, they also precisely forced the polymer to switch its position and conformation (Figure 11 a). [43] Fort his,t wo sets of so-called guiding strands were employed that allow the polymer to follow two different routes on the origami tile,depending on which type of guiding strand is applied. Theg uiding strands are also equipped with atoehold region-a short sequence of nucleotides which does not take part in polymer hybridization. Hence,t he guiding strands can be trapped by af ully complementary remover strand, which leads to the release of the polymer.Bysubsequently adding the other set of guiding strands,the polymer can be routed along the second track on the origami. These events can be monitored by FRET between the polymer and arranged reporter dyes.I ts hould be noted that approximately only half the origami structures displayed well-aligned polymers (AFM) and that surface contamination after the conformation switch significantly harmed imaging.I na ne nsuing study,t he group aimed to investigate the interaction between two different types of conjugated polymers by making use of the unique addressability of DNAo rigami (Figure 11 b). [44] In addition to the above-mentioned APPV-DNAc opolymer,t hey similarly synthesized ap olyfluorene-DNAp endant (poly(F-DNA)). However,n oi nterpolymer energy transfer was observed on conjugating either polymer to the origami rectangle.T his might be caused by al ack of interpolymer contact in combination with interference from unbound polymers, which demonstrates the limits for the conjugation of intricate polymers.The polymers were directly co-localized by hybridization of the side chains for further investigations.
DNAo rigami is an emerging platform to direct the motion of various objects on the nanoscale;h owever,t he

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Minireviews 6226 www.angewandte.org movement of the attached objects is often "fuel-based", that is,e mploying strand displacement reactions of respectively designed oligonucleotides to break and create old and new bonds.T hus,p urification after each step is often necessary. Baumberg and co-workers developed aD NA origami flexor whose actuation is mediated by at hermoresponsive polymer which can be stimulated externally ( Figure 12). [39] They designed af lexible DNAo rigami hinge structure, whereby poly(N-isopropylacrylamide) (PNIPAM) was attached on either side of the hinge.I nt his way,aP NIPAM-DNAconjugate was formed by catalyst-free strain-promoted cycloaddition and attached to the complementary capture strands within the hinge region. By fixing agold nanoparticle and afluorescent dye at opposite ends,the switch between the opened and closed state of the hinge could be optically monitored. Upon heating above the lower critical solution temperature (LCST) of 32 8 8C, PNIMPM becomes hydrophobic and forces the hinge to close.T his could be conclusively tracked by an increase in fluorescence as well as changes in the size distribution (DLS). However,t he AFM images obtained are av ivid example of how difficult direct visualization of conformation-altering DNAo rigami structures can be.
Tokura et al. further developed their surface-initiated ATRP on aDNA origami tile by transferring the technique to a3Dtube,preliminary paving the way towards 3D engineering of nanomaterials ( Figure 13). [45] Thea uthors designed asystem where orthogonal polymer growth is feasible:After coating of the outer surface with cross-linked PEGMEMA, the inner cavity of the origami tube was equipped with DNAzymes to induce the polymerization of dopamine. Whereas AFM images captured after the first polymerization step could reveal an increase in the height profile and, thus, the presence of polymer,n oi maging was possible of the polymer synthesized in the inside.The formation of polydopamine could only by monitored by absorbance spectroscopy, once more emphasizing how complicated the characterization of polymer-DNAh ybrid objects is.

Summary and Outlook
Thecombination of DNAorigami and polymers is astrong and emerging tool towards precise surface modification and the creation of elusively defined nanostructures in the low nanometer regime,t hus,r epresenting ak ind of a" top-up" approach that merges conventional bottom-up and top-down techniques.T odate,the arrangement of polymeric objects in av irtually infinite variety of geometries with precision of afew nanometers is not reported by any other methodology. It thereby pushes the limits of established lithography and self-assembly approaches by programming distinct nanodevices.F urthermore,D NA origami allows orthogonal decoration of polymers and other molecules,t hereby enabling the investigation of energy-transfer processes,a sw ell as the Figure 11. A) Switchingofapolymer strand conformationo naDNA origami by adding guiding and remover strands. B) p(F-DNA) (blue) and p(PPV-DNA)( green) on aDNA origami tile with the AFM image (right). Reprinted from Refs. [43,44] with permission.C opyright (2020) American Chemical Society.  installation of suitable reporter systems or targeting groups. In principal, two different strategies lead to the formation of such hybrid structures:either the polymer is grown from the DNAo rigami template or ap reformed polymer is linked to the DNAp latform. With regard to the studies discussed herein, it turns out that there are significantly more reports within the latter category.T he grafting of polymers from the origami surface is very challenging due to the extremely low concentration of DNAo bjects and the,t herefore,s mall number of initiator sites as well as the increased sensitivity to oxygen present because of the ultralow reaction volumes. Moreover,i ti sn ot possible to determine average molecular weights and distributions of the grown chains.F urthermore, the attachment of polymers to DNAo rigami faces some hurdles:t he solubility of the polymer is preferred to be compatible with DNA, and the entanglement of polymers and the folding of DNAm ight shield their reactive centers. However,t his strategy allows the larger scale synthesis of polymers and their thorough characterization prior to DNA origami fusion.
Whereas the electrostatic coating of DNAnanostructures with polycations may be considered as straightforward, it often only aims to stabilize the inherently susceptible DNA construct in biologically relevant media, but does not exploit the addressability of the platform to achieve molecular patterning.T herefore,hybridization of respectively modified polymers to complementary capture strands on the DNA origami is more expedient, but the conjugation efficiency and the grafting density is often reported as rather low.W eregard it as important to once again emphasize the characterization challenges which come along with the synthesis of polymer-DNAo rigami hybrid structures and which hinder fast progress in the field. Theu ltrasmall quantities of DNA origami hamper typical polymer analysis methods such as SEC,N MR, or DLS.T om onitor the impact of polymers on DNAo rigami at aq ualitative level, agarose gel electrophoresis can be employed. However,t he integrity of the structures cannot be confirmed in this way.T herefore, imaging techniques such as AFM and TEM have to be performed to visualize the objects.A sac onsequence of the small size of DNAo rigami, such techniques have to be operated in high-resolution modii, and sample preparation, for example,drying effects,has to be taken into account. The most representative image might be captured by performing AFM in al iquid environment, which corresponds to the natural occurrence of DNAo rigami in aqueous solution. Thus,i ndirect characterization, for example,F RET,c an also be utilized to monitor conformation changes.
In conclusion, the fusion of polymers with DNAo rigami holds great potential for designing programmable nanodevices with highest structural precision, and there are already pioneering investigations towards the application of this class of new materials.