DNA nanotechnologyhas seen large developments over the last 30years through the combination of solid phase synthesis and the discoveryof DNA nanostructures. Solid phase synthesis has facilitated the availabilityof short DNA sequences and the expansion of the DNA toolbox to increasethe chemical functionalities afforded on DNA, which in turn enabledthe conception and synthesis of sophisticated and complex 2D and 3Dnanostructures. In parallel, polymer science has developed severalpolymerization approaches to build di- and triblock copolymers bearinghydrophilic, hydrophobic, and amphiphilic properties. By bringingtogether these two emerging technologies, complementary propertiesof both materials have been explored; for example, the synthesis ofamphiphilic DNA–polymer conjugates has enabled the productionof several nanostructures, such as spherical and rod-like micelles.Through both the DNA and polymer parts, stimuli-responsiveness canbe instilled. Nanostructures have consequently been developed withresponsive structural changes to physical properties, such as pH andtemperature, as well as short DNA through competitive complementarybinding. These responsive changes have enabled the application ofDNA–polymer conjugates in biomedical applications includingdrug delivery. This review discusses the progress of DNA–polymerconjugates, exploring the synthetic routes and state-of-the-art applicationsafforded through the combination of nucleic acids and synthetic polymers. Within this massivecovalent structure twinned supramolecularly by its complementary sequence,the central dogma of biology operates with unrivalled precision thatfeatures nature’s evolutionary prowess. Chemically speaking,the genetic code is a set of colossal chains of DNA in which the diversityof life is governed through the sequence information stored withinthe DNA nucleobases .Although its biological role and impact are clearly unambiguous,DNA has a different facade in the synthetic world—collectivelyknown as DNA nanotechnology. Taking advantage of how the alignmentof nucleotides can be woven differently with multiple intersectingchains not present in nature, nanoscale structures can be tailoredwith near limitless geometric possibilities. From straightforwardshapes such as Y-shaped DNA-crossovers and multiarm Holliday junctionsto complex folding technologies such as DNA origami, these platformshave made revolutionary advances in biophysics, photonics, nanomedicine,and materials science. This is primarily due to how DNA architecturesgrant the capability to position two or more (macro)molecules/nanoparticlesof interest within a designated 3D space and orientation at nanometerresolution. The level of precision, coupled with the ease of DNA hybridizationmethods, has resulted in their widespread accessibility across alldisciplines.2+/Mg2+ has been a glaring limitation to its potential. As such,significant attempts to stabilize DNA structures involving the conjugationof polymers, hydrophobic molecules, nanoparticles, or even higherordered DNA weaving strategies have been achieved to protect the DNAphosphodiester bonds from hydrolysis. Interestingly, these approachesvery often result in the creation of novel materials with unique characteristicsand structures due to the differences between the physical propertiesof the DNA and its attached motif. Naturally, higher ordered architecturesresulting from hydrophilic/hydrophobic interactions are among themost abundant, with morphologies including micelles, vesicles, andtubes. The dimensionality of structures from 1D to 3D can be customizedby increasing the complexity of the DNA component, i.e. from singlestranded DNA (ssDNA) to multiarm double stranded DNA (dsDNA) to space-fillingDNA origami. By exploring the influences of synthetic (macro)moleculeson a non-natural, yet geometrically precise object, exclusive lessonson self-assembly, patterning, and interactions across 3D space canbe learnt.Nonetheless, while DNA-based technologies receivetheir deservedaccolades within the scientific community, its relatively poor stabilityand restriction toward aqueous medium containing CaIn this respect, polymer chemistry plays a crucialrole in conferringadditional properties to the already broad repertoire of capabilitiesdemonstrated by DNA. Here, the near limitless capacity for monomerdesign coupled with recent advances in radical polymerization methodologiesunder mild aqueous conditions offers a fertile avenue for the developmentof novel polymer–DNA conjugates in years to come. Hence, onecan easily envision the overwhelming extent of possibilities fusingpolymer-based technologies, i.e. block copolymers, sequence definedpolymers, and immolative polymers with DNA engineering.Furthermore,the influence of DNA technology on synthetic chemistryis not solely limited on the nanoscale. By mimicking how nature usesDNA as a template for the proliferation of life, synthetic moleculescan be designed to assemble similarly along a chain of ssDNA therebytransferring the sequence information provided by the template DNAonto the newly formed synthetic polymer chain. Beyond the recruitmentof small molecules or polymer precursors based on the recognitionof the nucleobases, DNA can be used to template polymer synthesisby functioning as a reactive center either as an initiator or a catalyst.In general, each part of the DNA—the nucleobases, the negativelycharged phosphate-deoxyribose backbone, the major/minor grooves ofthe double helix, as well as the 5′/3′ termini—isan attractive resource. Exploited differently, these parts of theDNA have expanded the breadth of polymer chemistry and provided alternativeroutes to fabricate nanoscale architectures.21 In this section we will focus on the chemistriesrelevant to the synthesis of DNA applicable to DNA–polymerconjugation. Specifically, we will discuss the possible techniquesto install reactive handles and the challenges to adapt each chemistryfor DNA synthesis. These functional handles can be divided into differentcategories where the target motif can be introduced through covalentmodifications or noncovalent interactions with the DNA structure  bead. TheCPG bead provides a high surface area to offer numerous attachmentpoints in addition to a high stability to chemical environments.5 Polystyrene (PS) beads can also be adopted for the solid phase approachoffering highly efficient synthesis at the nanomole scale.7 The solid phase synthesis method cycles through coupling, capping,oxidation, and deprotection steps for the addition of each nucleotide  a solid phase approach, employing phosphoramidite chemistry,is typically adopted. Phosphoramidite chemistry was first developedin the 1980s by Caruthers and co-workers and, through the optimizationand employment of a solid support, resulted in the high yielding automatedsystem used today.cleotide 2A. Once 8 Although modifications can be integrated at several positions onthe nucleotide, functional handles at the 3′- and 5′-endare most relevant to DNA–polymer synthesis for the productionof diblock copolymers. 5′-terminus-functionalized phosphoramiditesinclude reactive handles such as amines,9 carboxylic acids,10 alkynes,11 and thiols.12 Each functionalmoiety must be compatible with phosphoramidite chemistry and may alsorequire protection during the coupling process.Importantly, phosphoramidite chemistry is not limited to naturalnucleotides. Internal modifications can be incorporated through modifiedphosphoramidites as well as modifications at the 5′-end. Thechemical synthesis of ODNs is performed from 3′ to 5′;thus, 3′-end modifications are integrated through functionalizedsupports which the ODN chain can grow from. Modified phosphoramiditeswere developed alongside the described method producing varying nucleobase,sugar, and phosphate backbone moieties.There are severalprotective groups, including dimethoxytrityl(DMT) for amines and 2-chlorotrityl for carboxylic acids 2B, which14 A two-step reaction can conjugate the initiator group to the phosphoramiditemoiety, now available for solid phase attachment, followed by cleavageand deprotection in ammonia. This method provides an automated routeto synthesize ODNs bearing ATRP initiators. However, the attachmentof reversible addition–fragmentation chain transfer (RAFT)agents prior to deprotection and cleavage is not possible due to itsinstability in ammonia. Similarly, the norbornene-phosphoramiditeis also not available commercially; however, its synthesis and consequentincorporation has been established.15 Inthis case, two modified nucleoside phosphoramidites as well as the3′-functionalized column were synthesized demonstrating theversatility and ability to choose the position of the norbornene moiety.Modifications in the base pair region may not be optimal due to conformationdynamics,16 in addition to sterics andcharge repulsion from the overall DNA structure. Thus, to ensure thefunctional group is positioned externally  on the DNA structure, the 5-position on cytosineand the 4-O-position on thymidine were adopted forthe modification. These developments achieved through phosphoramiditechemistry have enabled the initial vision and future realization ofcovalent DNA–polymer synthesis.The incorporation of the functionalgroups described above intothe DNA makeup provides an avenue to synthesize DNA for conjugationto preformed polymers. Where polymerization directly from DNA is desired,the polymerization initiators, agents or monomers, must be attachedprior to polymerization. Atom transfer radical polymerization (ATRP)initiator phosphoramidites are not available commercially; however,several can be synthesized and have been incorporated through solidphase synthesis prior to deprotection and cleavage, demonstratinga feasible method to attach initiator moieties to ODNs.2.217 Many couplingreactions have now been demonstrated on functional handles, such asamines, thiols, and alkynes, which were previously incorporated duringsolid phase synthesis. As native DNA does not bear specific sitesfor chemoselective reactions, these compatible handles must be incorporatedprior to column cleavage through the phosphoramidite chemistry describedabove. The conjugation of these functional ODNs with small molecules has enabled the establishment of commonprocedures and reagents for coupling in the presence of unprotectedDNA.1 For a more efficient conjugationof DNA to polymers, several moieties are of interest that are notavailable as phosphoramidites for solid phase synthesis. For instance,norbornene–tetrazine chemistry was established as an efficientself-reporting method for DNA–polymer conjugation; therefore,the modification of a reactive ODN to bear these specialized functionswas desired.18 Both functional groups arenot available as a phosphoramidite commercially ; however, the synthesis in solution has beendemonstrated (18 The reactions were performed in adimethylformamide (DMF)–phosphate buffered saline (PBS) 1:1v/v solution to ensure solubility and stability of both the unprotectedODN bearing a carboxylic acid or N-hydroxysuccinimide(NHS) functional handle and the small molecules.18 In this case, the now adapted functional end-groups wereavailable for direct conjugation with a presynthesized polymer.For several functionalgroups, such as RAFT agents, the corresponding phosphoramidite iseither not commercially available or is not compatible with the solidphase synthesis process. However, the chemical handles available throughsolid phase synthesis can be postmodified after column cleavage toposition the unattainable groups. Although the chemistry itself issimpler than the synthesis of a phosphoramidite, unprotected DNA isa polyelectrolyte and requires an aqueous solvent system , which can present a new challenge. However,if organic solvents are required for the coupling reaction, surfactantscan be employed through complexation to mitigate DNA’s incompatibilitywith hydrophobic compounds.nstrated 3.18 The grafting from approach), the polymerizationinitiator or agent must be anchored to the DNA structure. Althoughthe synthesis of ODNs bearing ATRP initiators has been realized throughphosphoramidite chemistry, in contrast, RAFT agents cannot be conjugatedprior to the deprotection and cleavage steps. Postmodification cannottake place on the solid support and must be conducted in solutionafter cleavage. This synthesis was demonstrated through the postmodificationof amine DNA with NHS or pentafluorophenol (PFP) activated-RAFT agents,i.e. (((butylthio)carbonothioyl)thio)propanoic acid (BTPA) and 4-cyano-4-(phenylcarbonothioylthio)pentanoicacid (CPADB)  3.19 Such20 Although this approach hasnot been employed for DNA–polymer synthesis, efficient incorporationand subsequence coupling has been established,21 demonstrating an opportunity for alternative conjugationmethods with potentially improved yields and diversity.Additionally,in this section we have highlighted the approachesadopted for reported conjugations, which each require a functionalhandle from solid phase phosphoramidite synthesis. However, the functionalizationof DNA is not limited to this method. Chemical handles can also beincorporated through DNA polymerase extension with modified deoxynucleotidetriphosphates (dNTPs). The employment of modified dNTPs opens an alternativetoolbox to incorporate non-native functional groups through enzymaticsynthesis.2.3In addition to the portfolioof covalent chemistries available to the reactive groups of DNA, noncovalentapproaches exploiting the structural elements of DNA offer an alternativeroute for DNA functionalization. Native dsDNA is a highly chargedmolecule, formed through many noncovalent interactions which can beexploited for noncovalent complexation. ssDNA forms the duplex throughhydrogen bonding and van der Waals forces, π–πstacking, and hydrophobic effects in addition to the entropicallyfavorable disorder of water molecules. These interactions presentopportunities for noncovalent dynamic binding of small molecules tothe major and minor groove, between base pairs and to the phosphatebackbone 4.Throug22 forexample through the supramolecular assembly of DNA with the positivelycharged histone protein. These intrinsic interactions inspired theemployment of the phosphate backbone for DNA–polymer conjugatesynthesis. To afford this interaction, a reduction in ion–ionrepulsion is required where stabilization with Group 1 and 2 counterionsis commonly used. Thus, equally for the interaction with polymers,ion displacement must occur. A huge charge repulsion must be overcomein comparison to other biological molecules, such as proteins, whichare commonly neutral or have low charge counts. This is evidencedby the thermal energy required to bring two DNA molecules into proximitywhere electronic repulsion is 100× increased without the presenceof counterions.23 Due to the dynamic natureof DNA interactions, it is difficult to study the ion sphere to understandprecise interactions;23 however, the Poisson–Boltzmannequation can be employed to describe the relationship between a chargedmolecule and the counterions in solution to provide information aboutthe ion–ion interactions.25 Overall physicalproperties of the DNA–polymer product, such as zeta potentialand morphology, can also be analyzed to predict the complexation interactions.The interaction of polycationic polymers with DNA has gained interestdue to the increased ambition to deliver DNA to cells as potentialtherapeutics. The dissociation of DNA–polycations through theaddition of counterions can probe the effect of ionic strength onthe polymer interactions.26 Several counterionsof varying anion and cation units were added to DNA–polycationcomplexes, revealing the Group 2 ions, Ca2+ followed byMg2+, as the strongest dissociators in comparison to theGroup 1 ions. Anion competitors were also studied showing the largerand less electronegative I– caused the greatesteffect on polymer dissociation followed by Br–,Cl–, and F–.26 Conversely, the study of polymer binding has also beenperformed exposing an important note—the binding of cationicpolymers to DNA reduces the overall charge and thus alters the hydrophobicity.27 Therefore, balancing the concentration of cationicpolymer units to DNA’s anion charges has crucial implicationsfor solubility and aggregation. Full neutralization of charge leadsto DNA condensation, which, depending on the application desired,can have implications, such as steric hindrance of reactive sites.Similarly, the pH has large consequences on binding strength and,accordingly, the ability to form complexes.28 A lower pH can yield a higher degree of binding as observed by thesmaller and more tightly packed morphology in comparison to the largerstructures observed at a higher pH—a lower pH yields a higherextent of ionization.28 In addition tothe ion displacement, the shape of the polymer also has an effecton DNA complexation.29 The work of Tangand Szoka employed several polymers of similar molecular weight butvarying degrees of branching to investigate complexation with DNA.29 Interestingly, the unordered branched polyethyleneiminieyielded average complex diameters of 90 nm, which is approximately5% of the linear polylysine complex average diameter of 2000 nm. Thus,the shape can dictate both the polymer packing and the condensationof DNA. An understanding of the structure and charge effects of cationicpolymer binding to DNA can aid the design and choice of the respectivepolymer to avoid undesired structure deformation and to ensure applicabilityfor the desired function.The capability to employ electrostatic interactionswith the chargedbackbone generates a simple method for cationic molecules to bindto the sterically available anionic groups on DNA. The charged backboneplays many important roles in nature, such as guiding proteins andligands to designated positions,30 Many natural products have been discovered that offer native antibacterialor anticancer properties through groove binding and grant insightinto structural qualities appropriate for association.30 Through the desire to understand the interactivepockets, the precise interactions have been revealed and can thereforebe utilized for future therapeutic designs. Groove binders can targeteither the major or minor groove  and polyamides,31 thatare either cationic or neutral.32 Due tothe many interactions possible, binding is afforded through severalmechanisms. Specific interactions include H-bonding with the sugarC1, purine N3, and pyrimidine N1 as well as the base pairing moieties.32 Additionally, shape selective binding due tomolecular curvature is also apparent, where molecules match that ofthe native DNA structure.30 All these parametersbrought together lead to a high degree of target specificity. Largermolecules, such as proteins and carbohydrates, recognize and bindin the major groove. Although there are more donor and acceptor sitesin the major groove providing the platform for stronger overall enthalpicinteractions, fewer natural examples of major groove binders are described.33 Aminoglycosides are nonaromatic molecules whichpreferentially bind to the major groove of B-DNA due to the dimensionsand hydrogen bonding opportunities.31 Althoughinitial interactions may be with the phosphate backbone, studies employinga triplex DNA structure demonstrated the competitive release of thethird strand on the addition of an aminoglycoside dimer, implyingmajor groove binding of the aminoglycoside structure.35 A fundamental interaction is the protein–DNA dynamic bindingwith the major groove. In this case, the noncovalent H-bonds and saltbridges allow a reversible binding and release for processes, suchas transcription and gene regulation. The functional groups on thebases and ribose sugar provide several H-bond donor and acceptor sites.A detailed analysis of structure relationships has been reviewed previouslyby Thornton and co-workers.36 Althoughcurrent approaches to DNA–polymer conjugation do not directlyemploy groove binding, understanding the interactions will guide futuredesigns to improve polymer interactions through structure optimizationas well as positioning groups for functional anchors along the backbone.Proteins and aminoglycosides both offer many H-bonding sites in additionto positively charged residues to overcome repulsive forces. Throughthis knowledge, polymer design can be molded to encompass these attributes.However, it is important to also consider the structural distortionsgroove binding can have on the B-DNA structure. Groove binders thatpossess a strong overall binding enthalpy that outweighs the conformationalchanges can induce a fit.37 Depending onthe specific application of the DNA, these structural changes mayhinder downstream interactions.Groove binders have become a majortarget for small molecule andprotein binding for therapeutic action.r groove 4 through42 Natural productfunctions have consisted of several inhibitory roles which may actthrough allosteric interference of protein binding,43 influencing the development of anticancer drugs.44 Similar to groove binding, intercalators cancause conformational changes, such as extension. This extension isuseful to determine binding through length changes; however, it mayalso alter recognition and function of DNA as a genetic material.45 Intercalators, forming a mono- or bis-intercalationbetween one or over two base pairs, respectively,46 have been developed either for anticancer agents or asfluorescent dyes to visualize or quantify DNA.47 Several key features aid the association, such as a positive chargeas present on ethidium bromide .53 Therefore, attention to the multifaceted noncovalentdesign of DNA–polymer conjugates would increase binding strengthand thus has potential to prolong complex stability. The interactionsnoted for intercalator–conjugate assemblies lay the foundationfor intercalator–polymer design to guide the synthesis of precisepolymeric nanostructures.In the complexation interactionsdescribed above, each mechanismis explored individually; however, for several DNA binders, multipleinteractions are involved. A commonly adopted example is the combinationof intercalation and groove binding of antibiotics bearing peptidegroups which reside in the minor groove.3Polymerization was first noted in the 1800s andhas since developedto produce the synthetic polymers commonly used today, such as PSand Nylon 6. Due to3.1There have been large developmentsin the synthesis of covalent DNA–polymerconjugates; however, several limitations have hindered progress. Wewill first introduce the polymerization methods employed for DNA–polymerconjugate synthesis and highlight the limitations of these methodsin addition to the challenges of combining synthetic polymers withDNA. Through this discussion, we can build a greater understandingof the progress made in this field through solution- and platform-basedconjugation methods which are described in this section.3.1.166 Here, the equilibrium is determined by the rate of activation anddeactivation of the propagation reaction, where deactivation mustbe greater than activation to maintain a low concentration of radicalspecies. The first examples of ATRP required a metal catalyst, whichinitially led to developments involving reducing agents to reactivatethe metal center to reduce the required metal concentration; however,it could not be removed entirely. Metal free ATRP was later developedand employs an organic redox-active catalyst, therefore reducing thebiological toxicity of the reaction and increasing the compatibilityof ATRP for DNA conjugation.67 RAFT polymerizationwas developed shortly after ATRP and is also performed metal free.RAFT proceeds by a radical polymerization mechanism in the presenceof a chain transfer agent (CTA) to afford the necessary equilibriumfor reduced mass distribution . Grafting through is employed less frequently;however, it can efficiently synthesize many brush or hyperbranchedstructures. Nonetheless, each approach has drawbacks to either theyield or breadth of polymer conjugates achievable. These drawbackscan be accounted for by both the use of DNA in this system and alsothe polymerization conditions.The synthesis of covalently bound DNA–polymer conjugateshas seen large developments, now enabling the controlled synthesisof diblock copolymers consisting of many combinations of polymersand DNA nanostructures. The synthesis of DNA–polymer conjugatescan be categorized into three methods:  through 8. Grafti3.1.2grafting to) or in the polymerization reaction (grafting from). DNA is a highly ionic molecule requiringan aqueous environment which is readily compatible with hydrophilicmonomers and polymers; however, hydrophobic monomers and polymersrequire a solvent mixture to enable solubility. Organic solvents arecommonly poor liquids for DNA, altering hydrogen bonding, polarity,and hydrophobicity.69 Specifically, solventsconsisting of longer or alkyl-substituted chains cause the greatestdisruption.70 Consequently, initial studiesemploying the grafting to approach reported low yieldsfor the conjugation of hydrophobic polymers to DNA.71 However, as hydrophobic polymers also pose great interest,several groups have established improved methods such as DNA protectionwith counterions or sophisticated coupling chemistries.18 A thorough investigation into possible coupling reactions betweenDNA and poly(N-isopropylacrylamide) (pNIPAM) wasperformed by O’Reilly and Wilks.18 They found amine coupling and thiol–ene Michael additionreactions did not synthesize the correct product in organic solventsor were not reproducible. In each case, several solvents were trialedincluding DMF, dimethyl sulfoxide (DMSO), acetonitrile (ACN), andtetrahydrofuran (THF).Through the advancement of the polymerization methods describedabove, polymer synthesis, itself, is a highly established technique,which has been optimized for many monomer and polymer types. In parallel,the expansion of bioorthogonal chemistry has provided a plethora ofconjugation reactions between modified DNA and a variety of molecules,providing ample resources for DNA to polymer conjugation reactions.However, for DNA–polymer conjugates, there are several limitationsdue to the combination of these two materials in one reaction potbecause they can each provide contrasting properties. In the approachesdiscussed here, DNA is present either in the conjugation reaction can also overcome this challenge by thepolymerization of hydrophilic monomers to produce hydrophobic polymers.73 The use of PISA has been employed to successfully produce DNA–hydrophobicpolymer conjugates through the grafting from approach.74Similarly, the grafting from is deemed preferable to synthesize DNA origami–polymerconjugates as the steric hindrance is reduced.57 Steric effects are also a large consideration when couplingto a preformed polymer, i.e. grafting to. In thiscase, the larger polymers may shield the reactive handle and thereforereduce the reaction process.In addition to solvent compatibility,both blocks of the DNA–polymerconjugate are flexible polymers and can therefore shield the reactivemoiety. Steric effects are observed when coupling to all forms ofDNA—ss, ds, and nanostructures—although the effectsare different for the solution-based (ss and ds) and solid support(nanostructures and DNA origami) forms. Additionally, the sequenceof ssDNA requires a fine design to ensure the secondary structuresdo not hinder the reactive site. This also applies to dsDNA wherethe duplex may be in equilibrium with higher ordered structures. Inboth ss- and dsDNA, the sequence can be designed and modeled to ensurethat inhibitory secondary structures are avoided. Conjugation to DNAorigami presents the greatest hindrance for conjugation. The DNA origaminot only burdens the reaction center with steric hindrance, it also,where multiple sites are present on one structure, reduces the distributionof reaction sites in solution and requires a higher local concentrationon the origami. This causes drawbacks for both approaches; however, grafting to approach, the grafting from technique performs the polymerization in the presence of DNA, whichproduces additional challenges. When handling DNA, small volumes aretypically employed due to limited resources (reactive group-bearingoligos are commonly produced in microgram quantities); thus, when grafting from, small volumes are also adopted for the polymerizationprocess. This limitation is mainly apparent as both RAFT and ATRPtechniques are oxygen sensitive and therefore require an anaerobicenvironment. The approximate length of polymers can be controlledby the monomer to transfer agent or initiator ratio; however, oxygenis a radical scavenger and can therefore quench the initiated or transferredradical, altering the ratio. Radical polymerization in the absenceof DNA  can be performedin large volumes and is therefore not limited through the availabletechniques to remove oxygen. The most effective method to remove dissolvedoxygen is through N2 purging.75 N2 purging is possible in large scale synthesis; however, grafting from DNA is commonly performed in less than 300μL, preventing the efficient use of purging. Similarly, thefreeze–pump–thaw technique, whereby the solution isfrozen before a vacuum is applied to reduce the dissolved oxygen solubility,can take place in larger volumes, i.e. 1 mL. However, this techniqueis again problematic when performing the polymerization in small volumes,i.e. <300 μL, in the grafting from approachwhere the DNA concentration is limited. Volume loss may compromisereproducibility due to the effects residual oxygen will have on thepolymer length and yield. Additionally, DNA degradation can occurwhen the sample is subjected to repeated freezing and thawing—tensionforces are generated from ice crystals and may lead to strand breakage.76 An alternative method is enzyme degassing—atechnique that enables oxygen sensitive polymerization in air. Glucoseoxidase, an enzyme that converts oxygen to hydrogen peroxide, canperform successful enzyme degassing for RAFT polymerization in anopen, low volume vessel grafting from ODNs.77 Enzyme degassing provides an avenue to explorea wider range of polymers synthesized through the graftingfrom approach in the presence of DNA and in small volumes.However, purification to remove the enzyme is required after the reactionif downstream processes are desired. There are also other challengesassociated with the reduced concentrations available when workingwith DNA. Again, polymerization in isolation from DNA can be performedas optimized; however, when reactions with DNA for conjugation orpolymerizations from DNA are required, optimal concentrations maynot be possible with the limited amount of DNA (graftingfrom DNA origami. DNA origami is commonly synthesized inlow volumes (less than 100 μL) and in low concentrations (approximately50 nM). Polymerizations are optimal at mM concentrations; thus, toovercome this, sacrificial initiators are required in solution toensure the concentration limit is reached.57 Although this allows the reaction to proceed, polymerization alsotakes place in solution, adding competition to the DNA origami surfacepolymerization leading to downstream purification challenges.Although the limitations describedso far are mainly attained fromthe t of DNA 2. This i78 Sleiman,80 and O’Reilly,81 establishing bespokesequence polymerization. However, the intricacy and length of thesepolymers is still limited. Although there are several challenges whensynthesizing DNA–polymer conjugates, several groups have stillaccomplished many novel and innovative advancements which will bediscussed in the following sections.In addition tothe challenges described above, which originatefrom the physical and chemical environment, the absolute control ofthe polymerization method is still limited. Nature has the abilityto demonstrate sequence defined polymerization exhibiting controland self-assembly in a precise and reproducible manner. These characteristicshave inspired attempts to replicate controlled self-assembly withDNA–polymer conjugates. Several developments have been notedby the groups of Liu,3.1.3The development of conjugationchemistry has aided the increasedvariety of polymers conjugated to ODNs. In this section, we will describeconjugation reactions between free ODNs and polymers to synthesizea diblock product with a 1:1 ratio between each block, i.e. conjugationswhere the ODNs have been cleaved from the solid support prior to polymerconjugation. By performing the ODN cleavage prior to the conjugationreaction, a wider range of chemistries can be performed as deprotectionand side reactions are no longer limiting.88 Dueto the poor solubility of pNIPAM at high temperatures, the reactionwas performed at 4 °C to avoid precipitation in aqueous environments.Here, the reaction was performed in 10% DMF with borate pH 9.5, althougha successful reaction was also noted in 20% DMF with borate pH 8.2.89 In 2001, Park and co-workers employed similarchemistry to conjugate NHS-functionalized poly (PLGA) to amine-ODNs insolution.90 In water, PLGA degrades dueto its ester linkage; however, by adopting NHS-PEG, reaction in anaqueous system becomes possible.92 The comparison between these approaches highlightsthe challenge when conjugating DNA with hydrophobic polymers whichmay require organic solvents to dissolve. Additionally, the hydrophobicnature of the polymer may cause phase-separation from the ODN. Inanother example, Park and co-workers conjugated PEG to an ODN by amidecoupling. In this instance, an acid cleavable linker was incorporatedthrough an ethylenediamine intermediate attached between the ODN andthe tertiary amine group providing a route to DNA release in the acidicenvironments of cellular compartments.93 This demonstrates the potential for dynamic and changeable structureswhich will be discussed in 17 In the presence of the surfactant, DNA was soluble in DMF,DMSO, THF, and CHCl3 and provided the opportunity for higheryielding conjugation reactions toward hydrophobic polymers, such asPPO, PI, and PS. This approach therefore opens great potential foramphiphilic DNA–polymer conjugate synthesis in solution.One of the most directmethods of DNA–polymer conjugation employs amine-functionalizedODNs and NHS-activated polymers. Stayton and co-workers demonstratedsuccessful coupling of pNIPAM monofunctionalized with an NHS groupto a 7-carbon aliphatic amine-ODN.94 or a lactate95 groupwas present at the opposite end of the polymer to the acrylate groupbut both did not affect the reaction. A similar conjugation was performedby the same group; however, in this instance, the DNA was replacedwith RNA and the thiol group was positioned at the 5′-end incontrast to the 3′ as in the two previous examples. Here, thereaction was carried out with triphenylphosphine in DMF, which wasalso compatible and produced the desired product.96 Through the reactions with acrylamide described here, theincorporation of the acid labile ester group, β-thiopropionate,is consequently situated between the ODN and polymer blocks to enablea pH-responsive complex for RNA release. Herrmann and co-workers choseto perform Michael addition coupling with a maleimide activated PSto thiol-ODN. The maleimide-PS was dissolved in THF and mixed withthiol-ODN to result in a low yield of 13%.71Michael addition reactions have also been explored for ODN–polymerconjugation. Kataoka and co-workers synthesized a conjugate throughthe thiol–ene Michael addition of thiol-ODN to acrylate-PEGin tris-buffer pH 8.0 (aqueous). In each case, either an acetal97 Matyjaszewski andDas employed the polymer poly(oligo(ethylene oxide) methacrylate)(OEOMA), synthesized via ATRP of OEOMA to yield an average molecularweight of 14 700 Da, which was conjugated in high yields tothe desired ODNs. Here, ACN was adopted to stabilize Cu(I) in theabsence of a ligand while THF was added to dissolve the polymer. Conjugationsto PEG have also been demonstrated with moderate yields.98 However, to expand the diversity of polymerconjugates, conditions for amphiphilic conjugates are likewise desired.Reaction conditions were investigated by O’Reilly and co-workersfor pNIPAM in 100% DMF, with final yields between 70 and 90%.99 Hydrophobic polymer conjugation toward DNA wasdemonstrated using alkyne-modified poly(styrene) (Mn 4.4). In this case, the click reaction between PS andDNA produced high yields of 74% which had not previously been observedfor similar approaches, providing an improved avenue for connectingDNA with hydrophobic polymers. Matyjaszewski and Das also demonstratedthis click conjugation reaction with three polymers of similar molecularweight  to RNA.100 Here, the solvent was reduced to 0.6% ACN/H2O and couplingwas again successful. The versatility of DNA–polymer conjugatesynthesis was demonstrated by the click reaction on both RNA and DNAODNs with polymers of varying hydrophobicities, which opens opportunitiesfor downstream applications. Additionally, in each example, high yieldsare reported which exhibit a robust approach for conjugation in solutioncompared to the thiol–ene Michael addition reaction.In addition to amine and thiol anchors, azide- and propargyl-ODNcan also be exploited through the copper(I)-catalyzed Huisgen [3 +2] cycloaddition to conjugate free propargyl-DNA to azide-functionalizedpolymers in solution.18 This work was highlightedin 94 and higher concentrations, such as 25 μM,100 have been adopted. Therefore, where resourcesare not limited, successful conjugation via conventional couplingmethods can be envisaged.To address the challenge of poor yields often noted for DNA topolymer conjugation reactions in organic solvents, O’Reillyand Wilks conducted a comprehensive investigation of DNA–polymercovalent binding, analyzing amide coupling, thiol–ene Michaeladdition reactions, and tetrazene-norbornene coupling efficienciesto pNIPAM.grafting to approach allows the characterization of boththe polymer and DNA blocks to understand the composition and propertiesprior to conjugation. However, conjugation yields are often low dueto either solvent incompatibility, repulsion of charged polymers,or also the steric strain as discussed in grafting from approach can reduce the impact of steric straindue to consecutive single monomer attachments as well as increasethe ability to access shorter polymers blocks due to the ease of purificationof the final conjugate. Matyjaszewski and Das conducted the grafting from polymerization from DNA in solution and variedthe reaction time, catalyst, monomer, and salt concentration.13 Here, they polymerized OEOMA and showed thatat a high NaCl concentration of 300 mM, no polymer was produced andthat without salt, the higher molecular weight polymer was synthesized.Additionally, the lower Cu% (% compared to the monomer) yielded thelargest molecular weight along with a 120 min reaction time. Of note,Matyjaszewski and Das employed the activators generated by electrontransfer technique (AGET) of ATRP, which has been optimized for aqueousand biologically relevant reaction conditions.102 This advancement has been demonstrated by several groups and providesnew avenues for polymerization by grafting from ODNs methyl ether acrylate(OEGA), and oligoethylene glycol methacrylate (OEGMA), demonstratingthe versatility of this approach. Additionally, the length of thepolymer was controlled by the initiator to monomer ratio—ata ratio of 200:1 of monomer to RAFT agent, polymer length was 13.8kDa in comparison to 31.2 kDa at a ratio of 500:1. One significantchallenge of DNA–polymer conjugates is the incompatibilityof hydrophobic monomers or polymers with the hydrophilic DNA. However,with the methodology evolved from PISA, this incompatibility was exploitedto direct the formation of different DNA–polymer nanostructures.73 This technique was first demonstrated with ODNsby performing the grafting from using DMA, 4-acryloylmorpholine,2-hydroxyethyl acrylate, and OEGA.74 Restrictionsimposed by the DNA such as ultralow volumes and its associated problemswith degassing were circumvented by using glucose oxidase to ensurean oxygen-free environment for the polymerization.85 By adopting thermal RAFT polymerization and through the inclusionof enzyme degassing, the monomer to initiator ratio can be controlledprecisely and thus can allow the manipulation of architectures.74 In addition to thermal and photoinduced polymerizationmethods, ultrasonication is also a possible stimulus.86 Through the use of ultrasonication, room temperature andlow levels of Cu catalyst can be adopted to yield polymers with lowdispersity and high molecular weight.With the increased interest in synthesizing biologicallyrelevantpolymers and, therefore, the incorporation of biological material,methods to reduce the chance of downstream toxicity caused by contaminantsfrom the polymerization were desired. These contaminants typicallyresult from common ATRP methods which require a copper-based catalyst,free radical initiators, and reducing agents. However, through photoinducedATRP , free radical initiators and reducing agentsare no longer required and, additionally, the catalyst concentrationcan be reduced. Several acrylate- and methacrylate-based polymerswere synthesized by photoirradiation for 30 min under these mild reactionconditions,in situ to yield polypyrrole polymersgrown from and attached to DNA.87 Here,the polymerization is driven electrochemically and in solution toenable high chemical stability. This technique was also performedin the presence of noncomplementary and complementary ODNs demonstratingits capabilities to polymerize from both ss- and ds-ODNs.103Other polymerization methodssuch as those via oxidative approacheshave also been investigated to graft polymers from ODNs. The copolymerizationof pyrrole monomers present in solution and those conjugated to ODNwas performed 104 On binding, the complementarystrands bring the reactive monomers into close proximity for specificpolymerization reactions. A Wittig reaction was employed for simultaneouspropagation of the polymer and release of the monomer from its originalODN. To afford multiple cycling steps, the initial duplex exhibitsa short noncomplementary region (the toehold domain) to enable a fullycomplementary displacement strand to remove the propagation strandand leave the ss-ODN bearing the polymer chain. Although the localenvironment from each reaction step is constant, longer lengths arenot possible due to each reaction yield reducing cycled material.Beyond conventional homo and block copolymers,the attachment ofsequence defined polymers has made several interesting developments.In particular, sequence-specific polymerization of short polymerswas demonstrated by employing a cyclic binding and dissociation ofcomplementary ODNs (propagation strands) bearing the desired monomerfor sequential polymer growth.3.1.45000 and PEG10000 side chains.105 In their design, branchedPEG structures, named pacDNA (polymer assisted compaction DNA), weresynthesized via the consequential ROMP of norbornyl-NHS (N-NHS) andnorbornyl-PEG (N-PEG), for diblock synthesis, and through chain extensionROMP with N-NHS for triblock copolymer synthesis , again via ROMP prior to DNA conjugation to produce sphericalnucleic acids—macromolecular structures to be discussed in 110 A similar approach was adopted to synthesizeDNA–polymer conjugates where doxorubicin (DOX) was also covalentlybound within the structure.111 In eachcase, a diblock copolymer was synthesized by ROMP of norbornyl-DOXand N-PEG, where the carboxyl groups at PEG terminals were activatedwith EDC and NHS for 5 min prior to amide-coupling with amine-ODN.These two examples demonstrate the potential of DNA–polymerconjugates as drug delivery systems and their ability for high capacitydrug loading. Further details of applications will be discussed in 112 In this instance, the copper-free click chemistrywas performed in a 1:1 DMSO/DMF mixture in the absence of an aqueousbuffer. The examples described above demonstrate the employment ofdiblock copolymer structures synthesized prior to DNA conjugation.However, through the employment of a triblock copolymer, where eachblock initially consists of non-DNA content, controlled positioningof the ODNs along the polymer backbone can be realized. This was accomplishedthrough a triblock copolymer brush synthesized by the sequential ROMPof N-NHS, followed by N-PEG, and again N-NHS with Grubbs catalyst.113 Again, the NHS groups were then available tocouple amine-ODNs at the terminal polymer blocks of the brush structure.A further development in the design of triblock copolymers enabledthe synthesis of an N-NHS-N-PEG-norbornyl maleimide (N-MI) triblockand demonstrated a dual-ODN conjugation approach through the orthogonalreactions of amine-ODN to N-NHS and thiol-ODN to N-MI.113 This development enabled the incorporationof two distinct and specific ODNs within one nanostructure to openpossibilities for dual-functionalization.In thissection, the development of 1D structures, such as DNA–polymerbrushes will be outlined. The most common method described for DNA–polymerbrush synthesis is the ROMP of norbornyl bound to polymer side chainsand reactive handles, which can be employed for DNA attachment. Zhangand co-workers applied this approach to synthesize DNA–PEGconjugates consisting of PEGynthesis 9A.55,106l-glutamate N-carboxyanhydride114 to synthesize a polypeptide capable of clickchemistry between azide-functionalized ODNs and the propargyl groupafter the polymerization was complete.115 This technique enabled the synthesis of a hybrid peptide DNA brush,containing functional possibilities that have high biomedical relevancedue to its biocompatibility and postfunctionalization potential. Additionally,it provides a platform for high loading of DNA to target drug deliveryentities: 5–6 ssDNA molecules could be conjugated to one polypeptide.In an alternative approach toROMP, Liu and Li employed the ROPof γ-propargyl-in situ through initialmonomer conjugation and proceed with a grafting through type polymerization. This was demonstrated by the attachment ofnorbornyl to the ODN followed by ROMP (17 Dependingon the ODN length adopted (either a 7- or 14-mer), polymers of shortlengths  could be synthesizedand purified by polyacrylamide gel electrophoresis (PAGE). Of note,the ROMP here produced the longest polymer products in 100% THF. Asdescribed in graftingthrough strategy ensures that every monomer unit containsan ODN whereas a postpolymerization reaction is subjected to a statisticallydispersed functionalization of the side chains. This approach hasbeen similarly demonstrated with a peptide nucleic acid (PNA) wherethe PNA unit was covalently attached to a norbornyl group and subsequentlypolymerized via ROMP.116In each case described above, the ODN has been conjugated to thepolymer as a postpolymerization strategy. An alternative approachis to incorporate the ODN  by ROMP 9B.17 Dep117 In this instance, each “bristle”contained an azide group to afford click chemistry with an ODN.117RAFT polymerizationcan also be employed to synthesize 1D DNA–polymerconjugates. Martyjaszewski, Armitage, and Das adopted the RAFT polymerizationof methacrylate groups bearing macroinitiator side chains which canthen be polymerized to yield a “bottlebrush” polymer.graftingthrough, dual polymerization,can produce highly branched polymer structures. One example combinedself-condensing vinyl polymerization (SCVP) and cation ROP to producea poly(3-ethyl-3-oxetanemethanol)-PEO hyperbranched multiarm copolymer.118 An alternative approach replaced cation ROPwith RAFT polymerization to synthesize a hyperbranched polymer structureagain bearing PEG side chains but with a RAFT agent core containinga disulfide bond for redox-responsive drug delivery  ethyl acrylate which also served as theinimer to instigate branching. Once synthesized, a two-step substitutionwas performed to transform the chain end hydroxy groups to azide reactivegroups for click chemistry anchors. Copper free click chemistry wasthen performed utilizing a strained alkyne DBCO-modified DNA to affordhigh yielding conjugation in 100% DMSO. Through these methods, hyperbranchedstructures can be readily synthesized and postfunctionalized withthe ODN for the required drug loading.A similar approach to delivery 9C.119 Th3.1.5grafting to and grafting from methods.Platforms include nanomaterials, such as DNA origami, nanoparticles,and beads which together encompass metal, organic, and biologicalsurfaces consisting of varying 2D and 3D structures and properties (PtBA) and PS of molecular weights 3.9, 5.5, 8.5,and 14 kDa.125 The yield was determinedfor each conjugation to three ODN lengths  andrevealed an increase in yield with decreasing lengths of both polymerand ODN.125 This study highlights the limitationof steric hindrance on efficient conjugation where two flexible polymersare required to come into close contact for the reaction to occur.Conjugations with the lowest molecular weight PtBA and PS with the26-mer produced similar product yields to example click reactionsperformed in solution (between 70 and 90%);99 thus, either method can be adopted. However, on increasing the polymerlength to higher molecular weights, the yield decreased; for example,the yield was 56% for the conjugation between the 14 kDa PS and the26-mer on the CPG bead. Therefore, there is a trade-off between polymerand ODN length and product yield.An alternative approach is tosynthesize the ODN and polymer withcomplementary reactive click chemistry handles. Zhang and co-workerssynthesized several copolymers using azide-polymers with alkyne-functionalizedODNs on the CPG beads. Polymers consisted of poly weresimilarly performed.127 However, alterationsto relative equivalents were noted, 3× higher DIPEA and 4×less HBTU in comparison to the previous solid support reaction, demonstratingthat optimization is required for different reacting partners. Gianneschiand co-workers also synthesized an RNA–polymer conjugated followingthe same procedure for the aptamer DNA,128 demonstrating the robustness of this approach. Inspired from thedevelopment of conjugating polymers from the surface of CPG, presynthesizedpolymer nanoparticles, which bear chemical handles, became a naturalexpansion of the technology. A copolymer consisting of MA and azide-modifiedMMA units self-assembled into a nanoparticle, exhibiting the azideon the surface.129 Cu-free click reactionis then possible with DBCO-ODNs to yield 3D DNA–polymer nanoparticles.A combination of the solidphase synthesis approach with the presynthesizedpolymer brush was adopted by Gianneschi and co-workers to producea polymer brush with multiple ODNs attached to solid supports, followedby the deprotection and cleavage steps.grafting to approach, graftingfrom has been demonstrated on solid supports. Matyjaszewskiand Das explored polymerization from initiators bound to ODNs throughboth solid phase and solution phase ATRP by performing the polymerizationeither pre- or post-CPG bead cleavage.13 Performing the polymerization on the solid support provides easierpurification from the unreacted monomers and catalyst; however, theinitiator phosphoramidite must be compatible with deprotection andcleavage reactions. After the polymerization of OEOMA for 4 h, a molecularweight of 205 kDa was noted after cleavage from the CPG beads. Onsolid support, it is challenging to accurately quantify the concentrationof initiators and thus the initiator/monomer ratios. Conversely, insolution, concentrations can be determined and, therefore, optimizationsinvolving reagent ratios can be performed more accurately.Similar to the grafting from the ODN wasalso performed on a gold surface through the complementary bindingof a thiol-modified ODN (attached to the surface) to an ATRP initiator-modifiedODN.130 This approach produced poly(hydroxyethylmethacrylate) (PHEMA) from a gold surface, only when the complementaryinitiator sequence was bound. Further exploration employed a dual-functionalizedODN bearing a thiol group at the 3′-end and an ATRP initiatorat the 5′-end and demonstrated ATRP growth from a purely ssDNAsequence on the gold surface.131 Here,faster growth kinetics were observed when the initiator was presenton the ssDNA rather than directly on the surface. This effect canbe noted due to the localization of the Cu catalysts on DNA, increasingproximity to the initiator site. Several RAFT conditions were alsoinvestigated to optimize pOEGMA and PHEMA synthesis via RAFT polymerization.Temperature, time, CTA% surface density, and AIBN concentration wereeach explored and showed a temperature of 30 to 40 °C  is required for polymerizationto occur, a reduction in growth as AIBN concentration increases from0.4 mM, as well as a linear growth trend on increasing time for pOEGMA.83 Through the employment of a conductive 2D surface,the development of chips for applications requiring sensitive surfacescan be envisaged. To further establish the grafting from approach on a solid support, He and co-workers adopted a gold nanoparticleemploying the thiol–gold interaction.82 ATRP was performed through the initial coupling of an ATRP initiator,bromoisobutyryl, to the thiol-ODN, followed by Au nanoparticle (NP)attachment and incubation in polymerization reagents. Here, pOEGMAwas synthesized from the ODN-modified AuNP, and through the presenceof DNA, these reactions could be performed in an aqueous environmentwithout aggregation or solvent exchange requirements. In each casedescribed here, surface attachment is utilized; thus, characterizationof the polymer is limited and requires cleavage prior to analysis.Solidphase synthesis 80 Through the employment of phosphoramidite chemistry, thesequential addition of defined monomers via stepwise coupling andwashing was realized. In this instance, two phosphoramidite oligomersconsisting of either a hexaethylene glycol or hexaethylene (HE) unitwere adopted as hydrophilic and hydrophobic monomers and ordered ina controlled manor amine(THPTA) was adopted rather than CuI·P(OEt)3. However,as with the attempts using polymer brushes, steric effects are deterministicon the efficiency and can result in low coupling yields. By grafting from the nanostructure, steric restraints are reducedfor the monomer polymerization processes. The added advantage of employinga 3D DNA nanostructure backbone afforded polymer patterning throughthe site specific attachment.The examples so far have produceda conjugate containing DNA asa polymer block; however, they have not exploited the capabilitiesof DNA to guide the polymerization to precise assemblies through itssequence-specific interactions. DNA sequences can be programmed throughtheir specific base pairing to form folded nanostructures. DNA nanostructureswere first envisaged by Seeman in 1980, beginning as lattice structuresup to more recent examples of sophisticated DNA origami structuresas reviewed previously.d pNIPAM 11A. A 1057 The origami wasdesigned to exhibit “sticky” ssDNA at precise locationsto guide the initiator-ODN to the predesignated sites - and β-peptide backbones with various side-chainfunctionalities to accomplish longer and structurally more diversepolymers (26 kDa). Among several investigated conjugation strategies,copper-catalyzed alkyne–azide cycloaddition of AA-/BB-substratesproved to be most efficient. Moreover, by equipping the codon witha disulfide bridge between the polymer building block and the PNAadapter as a cleavable linker, the polymeric product could be liberatedafterward. Though the approach described here utilizes Watson–Crickbase pairing to produce sequence-controlled polymers without the needfor Nature’s enzymatic toolbox, the structural diversity ofthe substrates is still limited to macrocycles for entropic reasons.However, the codon design theoretically supports the incorporationof several building blocks without the need to readjust the templaterecognition site.One of the greatest advantages of DNA over syntheticpolymers is the unprecedented level of sequence-control as well asthe consequential precision in molecular weight and distribution.It therefore is attractive to exploit this unique characteristic andpotentially transfer the molecular information onto polymers. In thisway, ODNs can function as molecular matrices that recognize and interactwith guest molecules and, thus, organize them according to their sequenceand guide subsequent polymerization. An early example of how the sequenceof nucleic acids might be harnessed was demonstrated by Liu and co-workersehydes78 12A. Therehydes78 12B.59 Th137 Step-growth polymerizationtechniques typically suffer from poor control over molecular weightwhich inevitably leads to broad molecular weight distributions. Theytherefore employed nucleobase recognition to surpass these barriersand to synthesize conducting polymers of low dispersities. Insteadof using pure ODNs as a template, a thymine-decorated polymer wasmanufactured via living ring-opening metathesis polymerization (ROMP).Alignment of adenine-containing monomers by complementary base pairingalong the template strand and subsequent Sonogashira coupling affordedwell-defined daughter strands of conducting polymers. Whereas nontemplatedpolymerization or polymerization with an incorrect template only producedlow polymerization degrees and high dispersities (PDI > 2), thepresenceof the correct template significantly narrowed the molecular weightdistribution (PDI = 1.2) and yielded similar polymerization degreescompared to the parent strands. Thus, the all-synthetic strategy provedto be capable of programming the structure, length, and dispersityof commonly poorly-defined polymers by hydrogen bonding interactions.Following this, O’Reilly and co-workers furthered nucleobase-promotedpolymer templating by combining the methodology with a segregationstrategy that makes use of block copolymer self-assembly  and the thymine analogue1-(vinylbenzyl)thymine (VBT), PSt115-b-PVBT18, was synthesized that forms stable micelles inchloroform. The hydrogen bond interaction of the thymine templatewith a vinyl derivative of adenine (VBA) ensured the solubility ofthe adenine monomer in the solvent. Furthermore, the addition of thecomplementary adenine monomer led to a dynamic exchange of adenine-loadedtemplates into the micelle where the ensuing polymerization was takingplace. A so-called “hopping” mechanism of propagatingradicals along adjacent templates in the micelle core can theoreticallyexplain the remarkably high molecular weights of the daughter polymersof up to 400 kDa, even though the template only counts 18 thymineresidues. Hence, nucleobase templating enriched the free radical polymerizationto yield narrowly distributed daughter strands (PDI ≤ 1.08)by suppressing bimolecular termination in a confined environment.Another seminal approach to adoptfrom Nature’s capabilityto make exact copies of nucleic acid strands was investigated by theSleiman group.assembly 12C.138 T139 Here, RAFT polymerization of various acrylatesyielded polyacrylates with tunable side chains. As a key step, thesecopolymers were equipped with triaminotriazine handles by amidation of NHS moieties along the backbone. Melaminecan recognize thymine and uracil hydrogen bonding patterns in variousmedia and, therefore, ensured the hybridization with ODNs comprisingtwo blocks of the respective amino acid that are bridged by a cytosinelinker (dT10C10T10). Notably, theRAFT copolymers are intrinsically characterized by stereoregio backboneheterogeneity and still engage T/U rich ODNs with nanomolar affinityupon mixing in a 1:1 ratio. The supposed triplex hairpin binding modelof the compounds was further affirmed by FRET studies.The examples discussed so far mainly report on nucleobase-modifiedpolymers or closely related structures such as peptide nucleic acids.To further develop the field, Zhou et al. studied the triplex hybridizationof a polymer with a full carbon backbone alongside DNA and RNA ODNsto produce conjugates that might be suitable for DNA loading ontonanoparticles or delivery of siRNA in biomedical applications.140 The cationic polymer can entercellular membranes via charged-mediated endocytosis and successfullydeliver its cargo, thus inducing gene silencing. Supramolecular complexesof nucleic acids with cationic polymers have emerged prominently inthe area of gene delivery in order to circumvent viral delivery vectors.In this respect, several positively charged polymers such as polyethylenimine(PEI), poly l-lysine (PLL), polyvinylamine (PVA), and polyallylamine(PAA) are subjects of current research is commonly synthesized in a strongly acidic environment through chemicallyor electrochemically induced oxidation of aniline monomers. However,these harsh conditions prevent the use of biological templates asthey are highly sensitive materials. Oxidative polymerization of anilinetherefore necessitates the adjustment of reaction conditions towardmild pH ranges and tolerable oxidation agents  and hydrogen peroxide, photo-oxidationusing a ruthenium complex, and ammonium persulfate as an oxidant allproved to be capable of yielding PANI-decorated DNA conjugates. Ina similar approach, the group of He aimed to fabricate conductingpolyaniline nanowires along preoriented DNA templates which were alignedon a Si substrate.146 Oxidation of anilinewas also induced enzymatically by HRP and hydrogen peroxide. Adjustingthe pH value to pH 4.0 turned out to be crucial with regard to wirequality: at a pH of 5, the continuous formation of wires was interruptedby polyaniline particles; however, lowering the pH to 3.2 yieldedonly incomplete polymerization. Thus, the optimal pH range to ensurecontinuous and regular polymerization was on the one hand determinedby the optimized electrostatic alignment of aniline monomers alongthe template and on the other hand helped to retain sufficient enzymeactivity. By incorporating AuNPs into polyaniline nanowires derivedfrom DNA templates, Wang et al. showed the novel construction of hybridnanowires with expanded electrical properties.147 Therefore, a sequential assembly process was applied: positivelycharged AuNPs were aligned on surface-immobilized DNA templates, affordingnarrow AuNP chains. The gaps between neighboring particles were thenbridged by Ru-mediated photopolymerization of aniline derivativesin acidic media. The alternating AuNP–polyaniline hybrid nanowirecould then be visualized by atomic force microscopy (AFM).Likewise, electrostatic interactions can be further utilized totemplate polymerization along DNA, enabling these interactions todictate bond formation processes. There are a high number of studiesdemonstrating the use of DNA templates to exert control over the respectivesequence and structure as an appealing strategy in the field of conductingnanowires. In particular, polyaniline, polypyrrole, and polythiopheneare well investigated.n agents 13B. In a148 It could be shown thatmonomer functionalization had no negative impact on the oxidativepolymerization that was mediated through ferric chloride (FeCl3). Treatment of the conjugates with Tollen’s reagentled to the binding of silver cations to alkynyl residues which facilitatednucleation and growth of Ag clusters along the backbone. Comparedto unmodified poly(thienyl-pyrrole), many small nanocrystals are formedclosely to each other, attaining uniform distribution and enhancedconductive properties. In an ensuing study by Hannant et al., thesame monomer was employed to further investigate click chemistry forpostmodifications which might be of interest for sensing applications.150 Importantly, the pentynyl-substituted pyrrolederived nanowires retained structural integrity and remained active,i.e., conductive, after addition of azido molecules via the succeedingclick reaction. To broaden the monomer scope and to demonstrate thegenerality of the electrostatically driven templating approach, Houltonand co-workers polymerized dithienyl pyrrole monomers (TPT) alongDNA templates.151 Though this monomer onlycomprises 1/3 of the number of hydrogen donor sites compared to pyrroleand therefore reduced bonding capabilities, successful DNA recognitionand interaction was still possible. The higher structural regularityof the polymer justifies the use of a less active monomer, since simplymixing thiophene and pyrrole monomers only yields randomly alternatingsequences.Aswith many other aromatic heterocycles, pyrrole can also be polymerizedin oxidative environments. Thus, double helical DNA permits the constructionof 1D nanostructures through the organization of the pyrrole precursorsand subsequent oxidation and polymerization. In order to generatepure polypyrrole–DNA conjugates with alkynyl side groups, Horrocksand co-workers implemented chemical modifications into a thienyl-pyrrolemonomer (TP).149Investigation of the growth mechanism of pyrrolemonomers alongDNA templates by AFM imaging unveiled a stepwise polymerization process.First, low densities of conducting polymer bind to DNA as apparentlyspherical particles, followed by denser particle packing in a beads-on-a-stringfashion, which then resulted in subsequent dynamic reconfiguration,finally elongating and merging the particles in highly regular nanowireswith smooth morphology 13C.149152 Notably, the micelle formation of amphiphilesis not only determined by their concentration  but also significantly relies on the ratio of water phase toorganic phase, which is known as the critical water content (CWC).Based on the latter phenomenon, the group designed a self-assemblingsystem of polymers and DNA which is first guided by weak electrostaticinteractions that are subsequently caught up by hydrophobic drivingforces. They therefore utilized a copolymer that comprises two blocks,a hydrophilic PEG and a hydrophobic poly(4-vinylpyridine) (P4VP).Below the CWC for micellization, the positively charged P4VP interactswith DNA, forming linear complexes in which the DNA is encapsulatedby the polymer. Gradual increase of the water content allows for hydrophobicaggregation of the P4VP blocks between polymer chains in solutionand polymer chains on the DNA. The hydrophobic interaction then forcesrearrangement of the complex and finally leads to core–shellnanofibers in which DNA wraps around the hydrophobic polymer aggregate.When employing monodisperse and relatively short DNA templates, theseproperties were transferred into the DNA–polymer conjugateswhich are monodisperse in both length and width. The necessity ofthe DNA template is clearly evident since the copolymer alone onlyaccumulated in spherical micelles under identical conditions.The controllableinterplay of not only electrostatic but also hydrophobicinteractions between DNA and polymers opens up a completely differentpossibility to define the morphology of resulting conjugates. Oncemore, nature was used as a role model with respect to its outstandingability to store genomic DNA with the help of histones. Chen and co-workerstread new pathways for the noncovalent interaction of block copolymersand DNA by establishing a two-step self-assembly process.a priori synthesized polymer have to be equippedwith suitable intercalators. Although ethidium bromide is a very strongDNA binder, utilization of weaker intercalating molecules such asacridine can add the potential for reversibility to the complex. O’Reillyand co-workers employed RAFT polymerization to synthesize a seriesof acridine end-terminated polymers, including pNIPAM and pDMAm andinvestigated the effect of polymer structure on the nature and strengthof the interaction with DNA.40 Indeed,differences in complexation behavior were observed, which were potentiallycaused by the relative tendencies of the different polymers to self-assemblewhen brought into close proximity. For instance, a high load of pNIPAMonto calf thymus DNA and full occupancy of intercalation sites inducedirreversible aggregation. The DNA-guided vicinity of polymer chainsquasi-imitates the process when hydrogen bonds between the amide groupsof pNIPAM are formed, normally giving rise to its temperature-responsivecharacter. On the other hand, the compact structure of pDMAm toleratedhigher densities of polymer intercalation without aggregation occurring.Thus, the combination of pDMAm and a significantly shorter and well-definedDNA sequence (63 base pairs) yielded discrete and possibly brush-likenanoparticles with sizes of 10 nm. Importantly, DNA or polymer aloneas well as acridine-lacking polymer does not form comparable assemblies.In a different approach, Pike and co-workers instead used monomerswith π-stacking anchoring groups to arrange the monomers withinthe DNA helix and conducted polymerization after intercalation.51 Based on the intercalation of diazido derivativesof proflavine into the double helix, the azido groups exposed themselvesinto the major grooves of the DNA. Here, copper-catalyzed click reactionwith thienyl-pyrrole monomers was performed. Crucially, proflavineintercalation was not hampered by the ensuing click reaction of thefunctional groups nor was intercalation of a presynthesized unit ofintercalator and monomer successful, due to hydrophobic and stericimpediments. Polymerization of the spatially organized pyrrole unitswas initiated by residual oxygen species in the solvent, without theneed for a chemical oxidant.Besides the binding modes discussed here, the Watson–Crickbase pairing and resultant double helix structure further render DNAattractive for the intercalation of planar molecules. In duplex DNA,the environment of nucleobases leads to π–π-stackingof adjacent aromatic systems, a structural motif that has a greaterimpact on helix stability than hydrogen bonds of complementary bases.Compounds that recognize DNA via interaction within the stacking basesare therefore potential handles for attaching or growing polymersalong the DNA template. Hence, respective initiators, monomers, orthe 153 The polymer was synthesized by ATRP from ananthracene macroinitiator with 4 initiator sites, and DNA interactionwas induced by simple mixing of the compounds. Association constantsin the 105 M–1 range are higher thanthose found for intercalating molecules or electrostatic interactions,thus indicating successful groove binding. Furthermore, the associationstoichiometry was ascertained to be 1 polymer-adduct for every 5 basepairs, showing that most sites of DNA participate in the associationprocess. Although groove binding is mainly attributed to hydrophobicforces, van der Waals forces and hydrogen bonding may also be involvedin the process, promoted by the hydrophilic polymeric arms.While this strategy relies bothon intercalation within the stackingnucleobases and on chemical reactions taking place in the major grooveof DNA, DNA grooves alone also provide the opportunity for noncovalentattachment of polymers. Furthermore, the impact of adjacent base pairson the groove environment adds a certain level of sequence-specificityto the system which is less prominent among intercalators. To ensurean ideal interaction, groove binding compounds typically compriseat least two aromatic rings while still being flexible in contrastto rigid polycyclic planar molecules that are suitable for intercalation.Deiana et al. investigated the binding mode of an anthracenyl polymerwith dsDNA as well as the binding strength and mechanism.154 Supramolecular chemistry widely explores this class of host–guestsdue to their efficient and highly specific molecular recognition,low price, and simplistic modification. The spontaneously formed self-assembliesfrom the generated DNA-amphiphiles were found to be thermally stablewhich is attributed to extremely strong hydrophobic interactions.However, this technique is predominantly exploited to attach smallmolecules or oligomers rather than polymers.155 At this point it is important to note that this observation appliesto almost all approaches relying on noncovalent interactions. A rareexample for a polymer-based strategy is reported by Thelu et al. ina follow-up study to their work described above.156 Herein, adamantyl-terminated 8-arm PEG polymer is encapsulatedby X- or Y-shaped DNA carrying β-cyclodextrin at the end ofall ODN arms. The combination of a multivalent host with a star-likeguest led to nanogel formation, and the gelation was concentrationdependent. Moreover, the applicability of these nanoparticles in abiomedical context was accomplished by demonstrating successful drugloading, good cell permeability, and delivery into cells. Thus, dueto the universal and modular nature of the host–guest interaction,the approach holds the potential to be further developed.As already emphasized in the previous section (3.1), the synthesisof covalent amphiphilic conjugates of ODNs and polymers is problematic,especially due to solvent incompatibility, low conjugation yields,and phase-separation. Host–guest interactions can potentiallyalleviate some of these concerns by constructing a special hydrophobicmolecular environment to compensate for the difference between hydrophilicand hydrophobic components in solution. This environment exists ashydrophobic cavities within a general hydrophilic exterior thus allowingthe encapsulation of molecules that would otherwise phase-separate.Varghese and co-workers exploited this interaction mode by equippingDNA with a prominent host molecule (β-cyclodextrin) which cantrap adamantine-modified hydrophobic guests.3.2.2158 In addition,DNA origami’s integrity is highly dependent on sufficient levelsof divalent cations that allow close proximity of DNA strands by compensatingcharge repulsion of the phosphate backbones. Hence, several studiesseek to diminish DNA susceptibility through polymer-based approaches.Despite the drastically reduced accessibility of 2D and 3D DNA objectsin contrast to nonfolded DNA sequences, electrostatic interactioncan still be exploited in order to achieve an efficient polymer coating.Divalent cations are hereby substituted by polymeric polycations,either naturally derived or of artificial origin. However, withoutthe possibility of confining the electrostatic interactions in a designatedarea, coverage will occur nonspecifically which might hamper additionalpostmodifications.While ss-and dsDNA are extensively leveraged to tailor the polymersequence and nanostructure and to provide integrative functions, polymersthemselves can also benefit DNA in several ways. Higher ordered DNAarrays such as DNA origami are exceedingly versatile building platforms,but they face intrinsic stability drawbacks. Due to their nature,DNA objects are prone to enzymatic degradation through nucleases whenencountering physiological environments, thus limiting their progressin biomedicine. Even though degradation might be delayed by packingDNA structures of high density or by having multiple interstrand crossings,it cannot be completely impeded.60 Notably, for successful binding, the ratio ofamines within the polymer to phosphate groups within the DNA backbone(N/P ratio) was found to be pivotal. The number of cationic blocksof the copolymer; however, only had a minor impact on binding affinities.Ahmadi et al. followed suit and studied the effect of polymerizationdegree, charge density, and N/P ratio of linear PEI and chitosan oncoating efficiency.159 In line with otherstudies, the N/P ratio significantly determines the extent of interactionwith DNA. However, LPEI performed better than chitosan in protectingDNA from salt-depletion and nucleases, even at lower ratios whichthe authors attributed to its higher charge density. Moreover, thestudy reveals how challenging the characterization of polymer-decoratedDNA nanostructures might be. While bare origamis could be imaged bynegative stain TEM, LPEI-coated origamis were only visible after removingthe polymer shell. The indirect proof of polymer coverage describedhere is often the only way to analyze the conjugate (more examplesto follow within this section). In 2017, electrostatic coating ofDNA origami structures by copolymers from PEG- and polylysine-buildingblocks were reported by two different groups.160 The polylysine block interacts with DNA while the correspondingPEG block builds a protective layer and shields the sensitive DNAbackbone. The Schmidt lab synthesized PEG12 kDaPLys18 by ring-opening polymerization induced by an amino-terminatedPEG macroinitiator while the Shih group utilized commercially availablePEG5 kDaPLys10 polymers  and methacrylate derivatives (to serveas cationic blocks) were attached to a 60-helix bundled DNA structure.polymers 14A. Bothers  polymer while additional PEG side chains ensuredwater solubility of the construct which is required for successfulDNA conjugation later on. The sophisticated nature of the polymerled to a rather broad size distribution as characterized by GPC (340–3300kDa) and AFM (length range of 20 to 200 nm), presumably due to partialdegradation during purification. However, binding yields of polymersto various DNA origami tiles were still very high according to AFMimages. It should be noted that more complex alignments, for instance,a staircase path instead of a U-shape, lower the assembly efficiency.While the visualization of these 2D origami structures was accessibleby AFM, the same analysis was highly difficult for 3D objects. 3Dcharacterization was only possible when applying the DNA PAINT technique,again highlighting the challenges in investigating polymer–DNAorigami conjugates. In a follow-up study, the Gothelf lab furtherdeveloped their strategy by installing a programmable switch withinthe polymer configuration on DNA origami . Ding and co-workerstook inspiration from the latter mechanism and established a HRP-mimickingsystem to polymerize aniline on DNA.dopamine 15B.62 Novertime 15C.64 G-In the studiesreviewed here, the patterning of polymeric structureson a DNA template via noncovalent interactions remains relativelyscarce due to several aforementioned challenges. Complementary basepairing appears to be the most intuitive approach to arrange polymersalong several ODN anchors on a DNA template; however, it necessitatesa modification of both building blocks. Electrostatic coating leadsto nonspecific coverage in general, yet various groups designed DNAzyme-basedsystems to locally restrict polymer growth from the DNA surface. Thenanopatterning achieved using this method has allowed the customizationof structures in nanoscale resolutions that are yet unachievable byother technologies.3.33.3.1166 Here, a DNA nanocage was synthesizedbearing 8 ssDNA–amphiphile conjugates at the cube corners.The amphiphiles were found to self-assemble in the hollow cube core,demonstrating the guided self-assembly through DNA structures. Thepolymer consisted of HE and amino groups (Am) in the sequence 5′-Am-(HE)3-Am-(HE)3-Am-DNA-3′and was cross-linked with sebacic acid bis(N-succinimidyl)ester to produce the nanoparticle. Cross-linking the polymer chainsprovides the opportunity to alter the physical properties as wellas structure and function, which will be considered in In thesynthetic approaches reviewed above, we describe the conjugation ofpolymers to DNA where reactions are often compromised by the combinedlimitations of the polymer and the DNA. However, postfunctionalizationof the DNA–polymer conjugate offers further access to manipulatenanostructure behavior and function. To realize these postfunctionalizationprospects, functional groups need to be embedded in the polymer backboneor at the antipodal terminus. Depending on the polymer employed, modificationscan be implemented at points along the backbone; however, functionalgroups must be compatible with or protected from the coupling chemistryemployed for DNA conjugation. In doing so, there are several avenuesfor secondary polymer functionalization, such as cross-linking orsmall molecule attachment. Postconjugation polymer cross-linking ofan amphiphilic polymer was demonstrated to create a nanoparticle bearingsix ODNs.167 DNA coupling was performed through amine-NHS coupling,leaving the maleimide group available and unchanged. A thiol-modifiedfolic acid could then be coupled through a thiol Michael additionto yield a doubly conjugated polymer chain. Although there are a fewexamples in the literature, the development of biorothogonal reactionsdirectly fuels the available prospects of postconjugation functionalizationof DNA–polymer conjugates. In doing so, this strategy can potentiallyenable an increase in functional diversity as well as the capabilityto program complex solution behavior.Small molecular attachment is also possible through secondary couplingreactions. In one example, a PEG chain was functionalized by a NHSgroup at one terminal and a maleimide at the other.3.3.271 This PCR method employs presynthesized ODN–polymerconjugates as the primers, where the conjugation was produced via grafting to, to amplify a specific length of DNA. The amplificationoccurs through the binding of each primer to the long ssDNA sequence,followed by extension from the 3′-ODN-end by the DNA polymerase.This results in a long length of DNA bearing a polymer at the 5′-end.Monodispersity of the central block is therefore achieved and yieldseither a single or double terminal polymer conjugate determined bythe primers used. Additionally, as the primers are independent ofeach other, the conjugated polymer can be varied and therefore canbear two alternative polymers as part of the triblock, for example,PEG–DNA–PPO or NIPAM–DNA–PEG. In thisexample, amplification was employed to reshape the conjugate structure.Amplification can also pose benefits for downstream binding or signalenhancement. Rolling circle amplification is an alternative DNA replicationtechnique where DNA sequences are copied from circular DNA. In thecase of DNA–polymer conjugates, ssDNA penetrating from theconjugate is available to perform primer functions for DNA polymeraseextension from the 3-end. Specifically, ssDNA was bound to polymersat the 5′-end and could bind to the circular DNA at designatedpositions through complementary base pairing.168 Once bound, the DNA polymerase can perform the extensionand displacement for continued amplification resulting in long ssDNAprotruding from the polymer conjugate. Through the employment of postmodification,the long DNA chain is not present during the conjugation reaction,thus avoiding steric challenges. Therefore, the use of postcouplingextension to the DNA block through PCR and RCA demonstrates a syntheticapproach to long DNA–polymer conjugates. Chemistries couldalso be envisaged on DNA postcoupling. As with the polymer reactions,the compatibility with the DNA–polymer coupling reaction isrequired.Likewise,postconjugation modification can be achieved through the DNA block,offering several additional strategies unique to the DNA such as chainextension with PCR or hybridization of a complementary ssDNA. Onedrawback of DNA–polymer synthesis with a user-defined DNA sequenceis the length limitation of solid phase synthesis. The polymerasechain reaction (PCR) is a well-established technique employed to replicateshort (50 bp) to long (30 kb) lengths of DNA using specific primersto amplify the region of interest. In the case of DNA–polymerconjugates, PCR was employed to synthesize di- and triblock copolymerconjugates of defined DNA length and content bearing polymer termini.3.4grafting to approach as polymers can be fully characterized prior to attachment.For the grafting from approach, DLS and GPC86 are still applicable to determine the polymer length of 1D DNA–polymerconjugates; however, polymer characterization can pose greater challengeswhen 2D or 3D nanostructure conjugates are fabricated. Hence, for2D and 3D conjugates, AFM and high resolution transmission electronmicroscopy (TEM) can be used to determine the presence of the polymerconjugate on the DNA nanostructures. Spatial information in 2D/3Darchitectures of the polymer remains a challenge as it is often difficultto determine the orientation of the attached molecules. The overallstructure, such as a rodlike or spherical micelle, however, can bedetermined.74 Alterations to the DNA-blockcan also be analyzed by AFM.170 In one instance, theheight increase of the micelle due to dT incorporation by a terminaldeoxynucleotidyl transferase (TdT) polymerase could be monitored toassess DNA chain extension.170 Agaroseand PAGE are also useful techniques to confirm successful attachmentor polymerization through band shifts. PAGE is often the preferredmethod of electrophoresis due to the increased resolution and canbe employed to analyze covalent and noncovalent attachment. UsingPAGE, grafting from can be monitored by the appearanceof a new band at a higher molecular weight depicting successful polymerization,85 as can grafting to—the higher molecular weight band depicts efficient conjugation.99 A band shiftcan also be noted on noncovalent attachment due to the increase insize or also due to the change in overall charge—DNA coatedin positively charged polymers is either retarded or can even migratetoward the negative electrode. In addition, the formation of morecomplex nanostructures can also be analyzed by PAGE, where increasingmolecular weight leads to a band shift of lower migration.171 Both electrophoresis methods can be employedfor all DNA structures and preparation techniques; however, they donot show specifics, such as orientation or precise polymer length.Matrix assisted laser desorption/ionization spectroscopy (MALDI) canalso be employed to demonstrate that the correct molecular weighthas been achieved after conjugate synthesis via grafting to;91 however, conjugate flight can be challengingand not always achievable. The increase in experimental analysis techniqueshas enabled the analysis and therefore development of highly sophisticatedDNA–polymer conjugates, although further developments wouldenable absolute visualization or precision of the DNA–polymerconjugates to assist this field of research.The characterization of DNA–polymer conjugatesrequiresa range of techniques due to the variety of approaches and productssynthesized. In particular, the production scale of DNA–polymerconjugates has been a primary concern, which is often the bottleneckfor analytical tools with poor limit of detection. For conventionalpolymer synthesis, there are two key techniques: gel permeation chromatography(GPC) and dynamic light scattering (DLS), which are the benchmarkcharacterization techniques to determine polymer quality. For bothtechniques, approximately 10–50 nmoles of conjugate is requiredto provide an adequate signal for analysis. This often restricts thenumber of experimental variables one is able to explore due to thelimited amount of material available. GPC and DLS analysis can beapplied to all conjugates formed through the 4Supramolecularinteractions between biology and synthetic materialshave attracted more attention in recent years. Historically, the developmentof polymer and block copolymer assemblies has been widely regardedas the first entry of synthetic macromolecular objects in the understandingof the chain dynamics involved in complex solution processes. Biohybridsystems, in particular DNA–polymer conjugates, provide an intrinsicbridge to investigate supramolecular interactions promoted by therespective synthetic and biological entities. As expanded by previoussections, the chemical and physical orthogonality of both blocks allowsthem to be tailored largely independently, hence the potential fornanostructural design and supramolecular behavior to be studied ingreater detail.DNA–polymer conjugates behave like blockcopolymers, thusassembling into nanostructures due to hydrophobic and electrostaticinteractions. Moreover, the sequence-specific interactions betweenDNA strands allow programmed assembly of exquisite nanostructureswith stimuli-responsiveness, which is unique for block copolymerswith DNA components. Meanwhile, the polymer segments display diversechemical and physical properties, thus rendering more possibilitiesto tune the assembling behavior. Therefore, DNA–polymer conjugatescan result in large diverse self-assembled nanostructures, which isone of the most attractive features of such hybrids. In this section,we classify the DNA–polymer assembled nanostructures into staticand dynamic structures. The strategies to induce self-assembly, eitherby hydrophobic interactions through the polymer segment or by sequencehybridization by the DNA segments, are discussed for static structures,whereas the dynamic structures section mainly focuses on the stimulithat can trigger structural or behavioral changes.4.1There has beenmuch progress on the solution assembly of amphiphilic copolymers;however, the assembly of DNA–polymer conjugates has becomea promising new field due to significant breakthroughs in DNA conjugationchemistry. When DNA is attached to the polymer to obtain a DNA–polymerconjugate, the properties of the conjugate are dictated by both theDNA and polymer component. One of the most common approaches to synthesizeDNA–polymer nanostructures employs the hydrophobic interactionof polymers in combination with DNA sequence–specific recognition. 4.1.190 The formed micelles would continuously release ODNs by controllingthe degradation of PLGA chains and exhibited enhanced cellular uptakeby endocytosis thus leading to a new strategy for gene delivery. Sincethen, with the development of block copolymer self-assembly, DNA nanotechnologyand DNA–polymer micelles with different hydrophobic polymercores have been reported in succession.174Due to the wide range of hydrophilicand hydrophobic properties that can be customized by synthetic polymers,DNA–polymer conjugates consist of two categories: hydrophilicand amphiphilic DNA–polymer conjugates. Amphiphilic DNA–polymerconjugates can self-assemble into nanoconstructions with various morphologiesand are therefore attractive as unique nanomaterials. AmphiphilicDNA–polymer conjugates self-assemble into spherical micellesthrough the hydrophobic interactions with the aqueous solvent, exhibitinga hydrophilic DNA shell and a hydrophobic polymer core. The firstDNA–polymer micelles, formed from DNA–PLGA polymers,were introduced by Park’s group.b-DNA conjugates.121 The PS-b-DNA block copolymers were fabricated through CPG solid phase synthesis.The micelles subsequently formed by hydrophobic interactions of PS-b-DNA copolymers, and the resultant hydrophilic DNA exteriorof the micelles exhibited unique sequence-specific recognition properties.Furthermore, the average diameters of the micelle structures couldbe controlled by varying the DNA sequence length and PS molecularweight. Conversely, O’Reilly’s group employed copper-catalyzedazide–alkyne cycloaddition to synthesize the amphiphilic PS–DNAblock copolymer.99 The resulting amphiphilicPS–DNA conjugate could then form micellar structures in aqueoussolution. Whereas the conjugation efficiency achieved by Mirkin’sgroup was low, the copper-catalyzed azide–alkyne cycloadditionadopted by O’Reilly’s group resulted in PS–DNAconjugates in a 74% yield. The well-defined micelles were approximately20 nm in diameter in O’Reilly’s work, which confirmedthe amphiphilic properties of the PS–DNA block copolymer. Inspiredby the synthetic strategy of Mirkin’s group, Herrmann and co-workers122 constructed a new class of DNA–PPO micelles.Phosphoramidite-PPO derivatives were obtained by the reaction of hydroxyl-group-terminatedPPOs with phosphoramidite chloride. The corresponding derivativeswere attached to the 5′-end of ODN on a solid support to obtainthe DNA–PPO conjugates. The DNA–PPO conjugates self-assembledto form DNA–PPO micelles. Then, on the surface of the micellesseveral chemical reactions could be produced in a perfectly programmedand controlled manner. In addition to DNA amphiphiles, DNA triblockcopolymers can also be developed to assemble DNA–polymer micelles.Gauffre and Mirkin both prepared DNA–polymer micelles containingPCL hydrophobic cores by using triblock copolymers.174 The PCL hydrophobic cores could then be gradually degraded underphysiological conditions by cleavage of ester bonds with acid-promotionor esterase-catalysis.175 Gauffre et al.focused on micelle preparation and DNA-based recognition ability,174 whereas Mirkin’s group112 aimed to graft many more DNA strands onto the end of adiblock copolymer. Thereby they synthesized a micelle structure consistingof a DNA-brush block copolymer. The micelle exhibited a higher nucleicacids surface density, a higher melting temperature, and more effectivecellular uptake without transfection agent. In contrast, Caruso’sgroup176 prepared pNIPAM-cored DNA micellesin an alternative two-step approach. First, at a temperature lowerthan its lower critical solution temperature (LCST), the pNIPAM terpolymerformed micelle-like nanoparticles. Second, an amino-modified polyA30 or polyT30 was conjugated to the micelle-likenanoparticles to obtain DNA–pNIPAM micelles. Although therehave been some reports on the construction of DNA–polymer micellesusing DNA triblock copolymers, with the development of DNA amphiphilicpreparation technology in recent years, more work has been done toprepare DNA–polymer micelles using DNA amphiphiles. Sleimanand co-workers constructed HE–DNA micelles containing a polyethylenecore by the self-assembly of either HE6–DNA or HE12–DNA conjugates.80 A stepwisesolid phase approach was performed to sequence-specifically conjugatesynthetic HE oligomers on DNA, which would form monodisperse DNA–polymerconjugates with a defined sequence. The formed DNA–polymerconjugates displayed self-assembly behavior, which could be tunedby the polymer length employed. Specifically, in a buffer with 10mM Mg2+, HE–DNA conjugates containing five or fewerHE units existed as discrete molecules. In contrast, HE–DNAconjugates containing more than six monomer units could self-assembleto form DNA–polymer micelles. Furthermore, the successful preparationof the micelles was verified by encapsulating a guest molecule, NileRed, within the hydrophobic core of the micelles. Zhang’s groupalso constructed a DNA–polymer micelle through the self-assemblyof DNA–drug conjugates.177 By covalentlybinding nucleic acids and paclitaxel , theamphiphilic nucleic acid–drug conjugates could form micellarnanoparticles, which were structurally analogous to spherical nucleicacids (SNAs). This example treated the anticancer drug PTX as thehydrophobic core, which is discussed in more detail in Mirkin and co-workersprepared spherical DNA block copolymer micellescontaining a PS core through self-assembly of PS-178 dNTPs were sequentially introduced to the oligonucleotideprimer through the polymerase TdT. Thereby, ssDNA amphiphiles withhigh molecular weight and low polydispersity could be prepared bythe enzyme-catalyzed polymerization reaction in solution. Throughthe incorporation of multiple hydrophobic unnatural BODIPY fluorophore-modifieddUTP (B-dUTP) nucleotides at the terminus, ssDNA amphiphiles couldself-assemble into the starlike micelles. In this work, the successfulpreparation of micelles was verified by agarose gel electrophoresisand AFM and well predicted by the dissipative particle dynamics simulations.The enzyme polymerization method has great potential in the fieldof drug carrier development as the DNA building blocks can also beadopted as the drug.In additionto the series of methods reported above for preparingDNA–polymer micelles, Zauscher’s group used an enzyme-catalyzedpolymerization reaction to construct starlike micelles.180 fabricated helical ribbon structures -thiophene](PTOTT) with PEG-b-PTOTT.172 PEG-b-PTOTT drove the self-assembly which offeredan approach to attach the DNA’s molecular recognition propertiesto several self-assembling structures. In this work, not only DNA–polymernanoribbons were prepared, but also size-controllable DNA–polymervesicles by the assembly of DNA-b-PTOTT  (PPMA)-g-DNA nanofibers were fabricatedby Nardin’s group through the assembly of an amphiphilic poly(POX)-graft-DNA copolymer.182 Since POX is similar to a polypeptide in structure, it could beconsidered as a pseudo polypeptide. This was the first report of aDNA sequence and an assembly composed of a pseudo peptide througha nucleation polymerization mechanism. The nanofibrils were formedby inter- and intramolecular hydrogen bonding. Jiang et al. also successfullyprepared a primary nanofiber structure using the PPMA-g-DNA brush. The nanofiber structure possessed a hydrophilic DNA shelland a hydrophobic PPMA core formed by DNA strands covalently graftedto a PPMA backbone via “click” chemistry , a geometric structure that promoted theaggregation of proximal polymers and thus superamphiphiles, was henceselected for self-assembly of a micelle containing the cubes. Thenumber of aggregated DNA cages was programmed by changing the numberof hydrophobic polymer repeating units on the cages. With increasinglength of the polymer, the monomeric  structures gave way to a higher-order dispersed assemblyof the dimer and an increase in the finite number of aggregates, followedby a monodisperse oligomer micelle. Moreover, higher-order micelleassemblies were also possible by recognition of the external DNA nanostructuresto create functional macroscopic materials. A network of micelleswere formed by attaching different chains to the micelles of two differentpopulations and then adding a connecting chain , an approach to preparea series of shape-controlled DNA–polymer amphiphilic assemblies.191 This method could be employedto assist the assembly of polymers driven by the hydrophobic polymeroutside the frame, which offered greater control over self-assembly.First, they employed DNA-functionalized AuNPs as the frame to fabricatecustomized heterovesicles.190 Prepositioned,discontinuous leading hydrophobic groups (LHGs) were introduced byDNA hybridization to the corresponding positions of the frame, outliningthe edges of the designed structures. Other amphiphilic moleculescould be guided to fill the gaps between LHGs and ultimately producemonodispersed vesicles. This work improved the understanding of thefundamental mechanism of self-assembly through the FGA method. Subsequently,a similar method was employed to fabricate a 2D nanosheet and 3D heterovesicleassemblies by constructing DNA origami nanostructures.191 A variety of DNA origami shapes could be prepared through the foldingof a long scaffold DNA sequence by a series of carefully designedshort DNA strands. The ssDNA-modified amphiphilic molecule named DDOEGwas positioned reasonably on the DNA origami platform through sequentialspecific DNA hybridization to form a 2D hydrophobic framework domainon the DNA origami. Consequently, a homogeneous or heterogeneous 2Dnanosheet could be gradually formed on the DNA origami by absorbingadditional amphiphilic molecules into this hydrophobic frame domainthrough the hydrophobic interactions.191 Through this study it was finally demonstrated that the FGA strategycan overcome the obstacle of 2D amphiphile assembly in aqueous solution.Importantly, the size and shape of the 2D amphiphilic assemblies couldbe readily controlled by the shape of the DNA origami. Additionally,by varying the design of the DNA origami scaffolds, cuboid and dumbbell-shapedheterovesicles could be constructed.188 As shown in TDOEG (selected as the LHG) could be introduced to thesurface of the DNA origami through DNA hybridization. The added amphiphilemolecules, G2Cl-18 .98 Subsequent block copolymerassemblies were sequence-specifically and longitudinally positionedon robust DNA nanotubes. These materials were dynamic, and the blockcopolymer structures could also be cleanly removed from the DNA nanotubeswhen a specific competitor DNA sequence was added through constructing a thermally switchable triblock copolymer, DNA-b-pNIPAM-b-pMA  copolymerscurrently.202 PPO displays hydrophobic characteristics at room temperature andis hydrophilic at lower temperatures (below 20 °C). As a result,dynamic spherical micelles in aqueous solution can be formed by theself-assembly of ODN-b-PPO. Liu’s group hasmade outstanding contributions to the development of the thermallyresponsive DNA–PPO dynamic structures,196 in which they first fabricated DNA–PPO dynamic nanostructuresby the self-assembly of PPO-dsDNA-PPO triblock copolymers.195 The unimer of the inverse coil–rod–coiltriblock copolymer PPO-dsDNA-PPO was formed via specific DNA hybridizationbelow the LCST of PPO, which resulted in two thermally responsivePPO segments at the ends and a rigid dsDNA segment in the middle   wasintroduced into the micelles to achieve the pH-responsiveness (l-lysine) (PLL) and a lactosylatedPEG-antisense ODN (Lac-PEG–ODN) conjugate. An acid-labile linkage(b-propionate) was introduced into the micelle between PEG and ODNsegments to realize the pH-response (95 The experimentdemonstrated that the lactosylated PIC micelles exhibited an antisenseeffect (65% inhibition). However, the lactosylated PIC micelles withoutany acid-labile linkage exhibited a decrease in antisense effect (65→ 27% inhibition). This suggested the endosomal compartmentcontained a possible cleavage of the acid-labile linkage which wasin response to the lower pH. In a similar approach, Park’sgroup prepared pH-responsive polyelectrolyte complex micelles by thecombination of the peptide KALA and the ODN–PEG conjugates. This experiment mainlyillustrated that the micelles were more efficiently transported intocells than the c-myb ODN and the polyelectrolyte complex micelleswere a good carrier to deliver antisense ODN.Other than the facile adsorption to DNA via electrostatic interactionsto form pH-responsive DNA–polymer nanoparticles, DNA deliverycould also be achieved by conjugating DNA with polymers via a pH-responsivelinker. For instance, Kataoka and co-workers constructed pH-responsivepolyion complex (PIC) micelles,siveness 25A. Subsresponse 25B.95 Thnjugates25C.93 Th207 As shown in b-PPO conjugates were broughttogether and thus resulted in a triblock PPO–DNA–PPOcopolymer. In addition, by adjusting the pH to slightly basic conditionsthe “i-motif” structure could be decomposed into randomcoils. Meanwhile, DNA-b-PPO copolymers could embodythe characteristics of diblock copolymer under high pH. In this case,the assembly behavior of DNA-b-PPO was influencedby this stimuli-responsive nature. Hence, the morphologies of DNA-b-PPO dynamic nanostructures in an aqueous solution couldeventually be transformed by adjusting the pH and temperature stimulireversibly. DNA-b-PPO copolymers were assembled toform nanofibers at pH 5.0, and at pH 8.0 the morphologies would betransformed into spherical micelles small molecule drugs can be delivered by DNA–polymer conjugatesdue to the hydrophobic core of DNA–polymer micelles, and (2)polymers can be conjugated to DNA for stability enhancement of DNA–polymerconjugates.212 In this study, an SNA systemwas constructed to deliver the anticancer drug BKM120  for the treatment of chronic lymphocyticleukemia. The DNA–polymer conjugates (HE12–DNAconjugates) were prepared via solid phase synthesis and subsequentlyused to form spherical micellar DNA nanoparticles in aqueous solution.The hydrophobic HE12 core of the formed DNA nanoparticlesprovided a favorable environment to encapsulate the hydrophobic drugBKM120. Meanwhile, this work also showed that HeLa cells had enhanceduptake of these structures, as well as their cargo internalization.Moreover, in vitro studies illustrated that BKM120-loaded HE12–SNAs induced cellular death and apoptosis. In addition, Mirkin’sgroup used an extremely facile strategy to construct another new typesof polymer SNA conjugates which were comprised of PLGA NP cores thatcould be used to deliver small molecules ) block copolymer by electrostaticadsorption successfully enhanced the stability of DNA origami structures  block copolymerscoating around the DNA origami nanostructures, could protect the DNAstructures from nuclease digestion and denaturation. As a direct,economical, and reliable approach, this protection strategy couldbe used to protect various types of DNA origami nanostructures. Moreover,it was demonstrated that the DNA origami template directly determinedthe shape of the prepared DNA origami polyplex micelles after coatingwith PEG-p(lys) block copolymers. Shih and co-workers adopted a similarapproach to enhance the stability of DNA nanostructures. Through anoligolysine coating the major challenges which limited the effectiveuse of DNA nanostructures in vivo were overcome. in DNA) 29B. Howe5.2180 Through complementary base pairing, ssDNA chainsarranged along the edges of the formed ribbons were available to loadDNA-modified AuNPs, which served as a model cargo. Subsequently, Zhangand co-workers successfully realized the delivery of siRNA via nucleicacid hybridization.214 DNA-g-PCL brushes with complete water solubility were synthesized by graftingDNA onto a PCL trunk followed by the self-assembly of spherical andnanosized hydrogels by functional siRNAs cross-linking ethyl)disulfanyl)ethyl 4-cyano-4-(((propylthio)-carbonothioyl)-thio)-pentanoate-co-poly(ethylene glycol) methacrylate) (HPAEG) polymer witha backbone possessing a redox-responsive property was first successfullyprepared through combining RAFT polymerization and SCVP. Then HPAEGwas functionalized with AS1411 to form the drug delivery carrier (HPAEG-AS1411).Subsequently, the formed HPAEG-AS1411 nanoparticles were loaded withthe anticancer drug DOX  toform the assemblies and increase colloidal stability of this nanostructure.The model drug Nile Red was encapsulated into the core of the drugdelivery system which could undergo cleavage under UV irradiationby employing o-nitrobenzyl moieties.Several special shortssDNA sequences known as aptamers have beenshown to recognize cellular surface receptors and thus can be usedto import the desired DNA–polymer micelles into targeted cells.Zhu and co-workers fabricated a targeted drug delivery carrier bymodifying a polymer with the DNA aptamer AS1411.drug DOX 31B. HPAEOnce cleavagehad occurred, the hydrophobicity of the drug deliverysystem core was rapidly reduced, causing the breakdown of the systemand the resulting release of drugs 31C.221 dsDNA on the hydrophilic shell formed throughthe hybridization of the sgc8 aptamers were used to load DOX via intercalation,while the hydrophobic PLGA core was designed to encapsulate the PTXthrough hydrophobic interactions  micelles was synthesized through in situ rolling circle transcription(RCT), which promoted the generation of PLA poly shRNA microflowers.This was the first time to employ in situ RCT to produce a layer ofmultidrug resistance protein 1 (MDR1)-silencing poly shRNA concatemerson the DNA–polymer micelle. Hydrophobic DOX was concurrentlyloaded into the PLA cores, and then biocompatible and multifunctionalPEG-grafted polypeptides (PPT-g-PEG) were designedto induce microflowers electrostatically condensing into PLA-polyshRNA-PPT-g-PEG nanoparticles. The in vivo and invitro experiments finally revealed the great potential of this vectorin the combination of nucleic acid therapeutics and chemotherapeuticsin tumor therapy.In addition to DNA aptamers,short hairpin RNA (shRNA) was usedto design a targeted drug delivery carrier. Chen and co-workers constructeda new kind of nucleic acid–polymer nanodrug formulation whichcould be used to codeliver nucleic acid therapeutics (shRNA) and DOX32B.225 S177 Inthis work, the DNA corona of the SNA performed two functions: firstas a gene therapeutic and second as a delivery vehicle for small-moleculedrugs. A self-immolative disulfide linker could be introduced to thissystem to control the release of free drug. Multiple PTX moleculeswere combined to ssDNA to provide the sufficient driving force forDNA–PTX micelle formation through screening of the repulsiveinteractions between DNA strands.226 Theseself-assembled DNA–PTX nanostructures bypassed the need fora complex carrier system and allowed one to access a gene target anda drug target using only the payloads themselves. Additionally, asthese nanostructures enable gene therapy and chemotherapy using thepayloads themselves, cytotoxicity and immunogenicity challenges associatedwith complex vector systems are potentially avoided.The above work summarized that the DNA coronaof DNA–polymermicelles could be functional aptamers to increase the efficiency ofcell uptake. In addition, the DNA on the surface of the micelles canalso be used for gene therapy. As shown in Althoughsubstantial studies have been reported on DNA–polymerconjugates as drug carriers, their application is still limited bymany challenges. For instance, the stability of DNA nanostructuresneeds to be further improved to adapt to the complicated bioenvironment.To solve this challenge, scientists have attempted to use cationicpolymers to coat DNA nanostructures to improve their stability, asdescribed in 6The development of DNA technology, from chemical functionalizationto nanoscale strategies, has seen significant refinement and breakthroughin recent years. Given the ease of access toward these methodologies,the possibilities for which DNA can be exploited have expanded farbeyond conventional biology related disciplines. In particular, thisreview has summarized the impact of DNA on the construction of precisionmacromolecular conjugates and on programming supramolecular assemblies.On the molecular scale, DNA offers a high level of customizationprovided by both solid phase synthesis and state-of-the-art biorthogonalchemistry thereby granting general accessibility to the community.These chemical approaches subsequently inspired the development ofother reactions that can be conducted on the DNA such as polymerizationsand assembly driven chemistry. Nonetheless, the technical challengeof DNA stability, its polyelectrolytic nature, and nucleophilic functionalgroups are still prevalent issues. These considerations become morecomplex at the macromolecular level as conjugation toward hydrophobicpolymers/materials relies heavily on the exposure of reactive groupsthat are often masked by chain dynamics in solution. Nonetheless,the greater accessibility of DNA materials will ensure increasingefforts in method development.At the nanostructure level, supramolecularinteractions dictatedby both the polymer and the DNA component take the central role indetermining their eventual morphology. As such, polymer physics ofDNA–polymer materials has overwhelming room for future innovation.In this respect, DNA offers new insights in phase transitional behavior,packing of polymer chains, and crystallinity by using precise chainconformational switches such as i-motifs and hairpins. Additionally,the balance between the ordered and monodisperse sequences of DNAagainst the intrinsically disordered polymer chain is a unique relationshipthat can foster novel nanoscience frontiers. Customization at thissize regime involves polymer design and the effect of different monomers in directing structureformation. Hence, most technical challenges involve irreversible aggregationof the conjugates due to incompatibility between the two blocks andthe solvent system.grafting to and graftingfrom methods. Here, transference of the shape profile andinformation from the precise DNA scaffold to control polymerizationbecome a powerful technique to guide polymer synthesis and orientation.However, additional restrictions with regard to stabilizing ions areimposed as polygonal DNA structures are much more fragile. Increasingefforts by exploring DNA-crossover techniques have shown optimizticoutcomes, and its broader application can be envisioned. While thisreview covered the aspect of polymer stabilization of these polygonalobjects, it still lacks breakthrough strategies that allow broad implementations.Structures of higher complexity rely onDNA playing a larger rolein directing structure formation and hence are limited to aqueoussystems. This includes highly polygonal 3D objects created by theDNA origami technology where polymer chains can be attached at anyposition by both At each length scale, it is unambiguous how the synergy betweenDNA and macromolecular chemistry can bring about new horizons in multipledisciplines. However, at the same time, multiple challenges in eachfacet need to be overcome by the scientific community to access thisknowledge. As such, every success will bring forth new technologiesand features that will stimulate the collective understanding of precisenanoscopic 3D architectures in materials science and nanomedicine.