Nucleobase-Containing Polymers: Structure, Synthesis, and Applications

polymers

Review Nucleobase-Containing Polymers: Structure, Synthesis, and Applications

Haitao Yang 1 and Weixian Xi 2,3,* 1 School of Materials Science and Engineering, Nanchang Hangkong University, Nanchang 330063, China; [email protected] 2 Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA 90095, USA 3 Department of Orthopedic Surgery, University of California Los Angeles, Los Angeles, CA 90095, USA * Correspondence: [email protected]; Tel.: +1-303-748-3805

Received: 10 November 2017; Accepted: 27 November 2017; Published: 1 December 2017

Abstract: Nucleobase interactions play a fundamental role in biological functions, including transcription and translation. Natural nucleic acids like DNA are also widely implemented in material realm such as DNA guided self-assembly of nanomaterials. Inspired by that, polymer chemists have contributed phenomenal endeavors to mimic both the structures and functions of natural nucleic acids in synthetic polymers. Similar sequence-dependent responses were observed and employed in the self-assembly of these nucleobase-containing polymers. Here, the structures, synthetic approaches, and applications of nucleobase-containing polymers are highlighted and a brief look is taken at the future development of these polymers.

Keywords: nucleobase interactions; polymer synthesis; self-assembly

1. Introduction Nucleic acids in nature (DNA/RNA) represent the most powerful biomolecules. They serve as genetic material by hybridizing to a corresponding complementary strand to perform gene transcription and translation. Specifically, DNA carries genetic information from one generation to the next in living systems, and RNA guides the accurate assembly of amino acids into proteins by copying genetic information from DNA. Furthermore, in the materials science field, nucleic acids are also exploited for their selective hybridization capacities, unique geometric structures, ability to form nanoscale building blocks, directed self-assembly, and flexible programmability [1–4]. For example, DNA functionalized particles assemble into unique structures simply through the hybridization of functionalized DNA strands. This simple interaction also enables DNA nanostructures to fold into controlled shapes [5,6], nanoactuators [7,8], nanosensors [9–12], nanotweezers [13,14], and other nano-devices [15–18]. Additionally, DNA has been used as a therapeutic measure for various other biological purposes [15,16]. Despite the fact that DNA has so far enabled a handful of revolutionary advances for proof-of-concept nanomaterials, its broader impact has been limited due to restrictions imposed by the cost, scale, and scope of DNA. Thus, various attempts have been made by polymer chemists to move towards a DNA-mimicking nucleobase containing polymers [17–19]. In this review, we will highlight the recent advances in synthetic nucleobase-containing polymers, from their synthetic methods to their representative applications in materials science.

2. Nucleobase-Containing Polymers in Nature DNA and RNA are the nucleobase-containing polymers in nature. The central molecular interaction between these polymers is a hydrogen bond between corresponding nucleobases; these nitrogenous bases are adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

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DNAusually usually forms formsduplexes duplexes that are that stabilized are stabilized by hydrogen by hydrogen bonding bonding between between adenine adenine(A) and thymine (A) and thymine(T); cytosine (T); cytosine(C) and (C)guanine and guanine(G). RNA, (G). in RNA, most inca mostses, consists cases, consists one strand one strandof nucleic of nucleic acids, acids,using usinguracil uracil(U) instead (U) instead of thymine of thymine (T) in its (T) nucleobase in its nucleobase pairing [20]. pairing Besides [20 ].the Besides biosynthesis the biosynthesis of DNA/RNA of DNA/RNAin nature, oligonucleotide in nature, oligonucleotide synthesis is synthesisbased on isphosphoramidite based on phosphoramidite chemistry, which chemistry, was whichdeveloped was developedin the 1980s in theand 1980s later and combined later combined with solid-phase with solid-phase supports supports and and automation automation [21,22] [21,22 ](Figure (Figure 11).). However,However, thethe length length of syntheticof synthetic oligonucleotide oligonucleotide is limited is limited by the by coupling the coupling efficiency efficiency and the following and the purificationfollowing purification steps, which resultedsteps, which in a typical resulted length in ofa synthetictypical single-strandedlength of synthetic DNA/RNA single-stranded of around 70~100DNA/RNA bases, of witharound low 70~100 error ratesbases, [23 wi].th Most low recently,error rates Zauscher [23]. Most et al. recently, utilized Zauscher transferase-catalyzed et al. utilized livingtransferase-catalyzed chain-growth polycondensation living chain-growth to polycondensation synthesize well-defined to synthesize natural well-defined DNA sequences natural such DNA as homopolynucleotidessequences such as homopolynucleotides (poly(dT), poly(dA)), (poly(dT), randompolynucleotides, poly(dA)), random and polynucleotides, block polynucleotides and block with morepolynucleotides than 1000 bases. with This more work than is an1000 excellent bases. example This work of employing is an excellent enzymatic example living polymerizationof employing toenzymatic achieve DNAliving synthesis polymerization in the absence to achieve of aDNA DNA synthesis template in [24 the]. absence of a DNA template [24].

FigureFigure 1.1. GenericGeneric chemical chemical structures structures of of nucleobase-containing polymers polymers in nature and thethe phosphoramiditephosphoramidite DNADNA synthesissynthesis cycle.cycle.

3.3. Synthetic Nucleobase-Containing PolymersPolymers ToTo mimicmimic DNA/RNADNA/RNA sequences in nature and implementimplement their sequence recognition behaviorsbehaviors inin polymerspolymers chemistry chemistry and and materials materials science, science, synthetic synthetic chemists chemists have chosenhave chosen nucleobases nucleobases as a “smart” as a functional“smart” functional group in group the side in chainthe side of theirchain polymers. of their polymers. Furthermore, Furthermore, with the with development the development of various of polymervarious polymer synthesis synthesis skill sets, skill especially sets, especially controlled controlled living polymerization, living polymerization, more and more more efforts and more have beenefforts made have toward been made nucleobase-containing toward nucleobase-contain polymers synthesising polymers and manysynthesis of them and havemany been of them exploited have intobeen self-assembly exploited into polymeric self-assembly structures polymeric such structures as micelles such or as nanogels micelles basedor nanogels on speciﬁc based nucleobase on specific interactions.nucleobase interactions. In subsequent In subseq sections,uent we sections, will highlight we will differenthighlight nucleobase-containing different nucleobase-containing polymers basedpolymers on differentbased on polymerization different polymerization mechanisms mech andanisms discuss and representative discuss representative examples of eachexamples kind ofof nucleobase-containingeach kind of nucleobase-containing polymer in various polymer polymer in various science polymer applications. science applications. The common The methods common for preparationmethods for ofpreparation NB-containing of NB-containing polymers were polymers showed were in Table showed1. in Table 1.

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Table 1. Common methods for the preparation of NB-containing polymers.

Polymerization method NB-Monomer Length References Styrenes and derivatives Acrylates ATRP Methacrylates 10~100 bases [25–27] (Meth)acrylamides Miscellaneous monomers Styrenes and derivatives Acrylates RAFT 10~100 bases [28–32] Methacrylates (meth)acrylamides Styrenes and derivatives NMP 20~100 bases [33,34] Acrylates Conventional radical polymerization Common monomers 80~200 bases [35–37] Azides Alkynes Thiols Click chemistry [38–40] Vinyls 10~50 bases Acrylamides Dienes Cyclic monomers (e.g., cyclic Ring opening polymerization 10~40 bases [41–43] oleﬁns, acetals, lactams, lactones) Other polymerization Thiophenes and derivatives N.A. [44]

3.1. Synthesizing Nucleobase Polymers by ATRP Since Atom Transfer Radical Polymerization (ATRP) was independently discovered by Sawamoto and Matyjaszewski et al. [45,46] in 1995, by virtue of affording polymers of narrow molecular weight distributions, high molecular weight, and advanced macromolecular architectures, it presents a powerful method to prepare a great variety of polymers, including nucleobase-contained polymers. Spijker et al. utilized ATRP to synthesize a series of well-defined poly(ethylene glycol), (PEG)–poly(adenine) and PEG–poly(thymine) nucleobase containing block co-polymers from a PEG-based macroinitiator [26]. Nucleobase-functionalized monomer (thymine and/or adenine), catalyst (CuCl), ligand (bpy) and PEG-based macroinitiator (stoichiometric) were mixed and underwent typical ATRP procedures. The expected conversion was analyzed by 1H-NMR spectroscopy. Finally, the reaction was quenched by 1-phenyl-1-(trime-thylsiloxy)ethylene (showed in Scheme1). Furthermore, to demonstrate the unique properties of the nucleobases in the sidechain, they investigated the aggregation behavior of two complementary block copolymers in water (The structure of assembled copolymers is shown in Scheme1). They found an interaction between the complementary thymine and adenine moieties, which resulted in an increased solubility of the block copolymers by an improved shielding of the hydrophobic blocks. In 2007, the same group reported the synthesis and controlled polymerization of all four nucleobases’ (thymine, adenine, cytosine, and guanine) methacrylate monomers through ATRP method [25]. Technically, by alkylation of the nucleobases with 3-bromopropyl methacrylate, thymine-, adenine-, cytosine-, and guanine-functionalized methacrylate monomers were prepared and their ATRP kinetics were examined. Interestingly, they found the formation of guanine and cytosine monomer with copper(II), and these two specific polymerizations required more addition of catalyst and the use of PMDETA. From this study, all four unprotected nucleobase monomers could be polymerized in a controlled manner, but there is still space for improving the level of control in the synthesis of nucleobase-containing polymers through ATRP. Similarly, Cheng et al. [47] synthesized poly[1-(4-vinylbenzyl)thymine] (PVBT) through ATRP at a high temperature (showed in Scheme2) and, furthermore, performed nucleobase recognition through adding presynthesized 9-hexadecyladenine (A-C16) in the presence of polythymine homopolymers. Specifically, due to the hydrogen bonding interactions between the thymine groups of PVBT and Polymers 2017, 9, 666 3 of 25

Table 1. Common methods for the preparation of NB-containing polymers.

Polymerization method NB-Monomer Length References Styrenes and derivatives Acrylates ATRP Methacrylates 10~100 bases [25–27] (Meth)acrylamides Miscellaneous monomers Styrenes and derivatives Acrylates RAFT 10~100 bases [28–32] Methacrylates (meth)acrylamides Styrenes and derivatives NMP 20~100 bases [33,34] Acrylates Conventional radical polymerization Common monomers 80~200 bases [35–37] Azides Alkynes Thiols Click chemistry 10~50 bases [38–40] Vinyls Acrylamides Dienes Cyclic monomers (e.g., cyclic Ring opening polymerization 10~40 bases [41–43] olefins, acetals, lactams, lactones) Other polymerization Thiophenes and derivatives N.A. [44]

3.1. Synthesizing Nucleobase Polymers by ATRP Polymers 2017, 9, 666 4 of 24 Since Atom Transfer Radical Polymerization (ATRP) was independently discovered by Sawamoto and Matyjaszewski et al. [45,46] in 1995, by virtue of affording polymers of narrow the adeninemolecular group weight of A-C16,distributions, the high biocomplementary molecular weight, and PVBT/A-C16 advanced macromolecular hierarchical architectures, supramolecular it presents a powerful method to prepare a great variety of polymers, including nucleobase-contained complexespolymers. were formed Spijker et in al. dilute utilized DMSO ATRP to solution. synthesize In a series the bulk of well-defined state, these poly(ethylene complexes glycol), self-assemble into well-ordered(PEG)–poly(adenine) lamellar and structures; PEG–poly(thymine) these self-assembled nucleobase containing structures block co-polymers can be from tuned a PEG- by different A-C16 loadings.based macroinitiator In 2015, a[26]. water-soluble Nucleobase-functionalized triblock polymer monomer and(thymi ane pentablock-polymer and/or adenine), catalyst containing nucleobases(CuCl), were ligand synthesized (bpy) and by PEG-based Haddleton macroinitiat and Garciaor (stoichiometric) et al. [27]. Theywere mixed utilized and ATRP underwent to polymerize typical ATRP procedures. The expected conversion was analyzed by 1H-NMR spectroscopy. Finally, methacryloylthe uridinereaction andwas methacryloylquenched by 1-phenyl-1-(trime adenosine from-thylsiloxy)ethylene bromoisobutyroyl (showed poly(ethylene in Scheme glycol)1). (PEG) macroinitiators.Furthermore, Furthermore, to demonstrate they the investigated unique properti thees secondary of the nucleobases structures in the of sidechain, triblock polymersthey and pentablockinvestigated polymers the in wateraggregation by CD behavior spectrum of two and complementary mixing adenosine block copolymers and uridine in water. triblock (The copolymers in water resultedstructure inof assembled a new supermolecular copolymers is shown self-assembled in Scheme 1.) They structures, found an which interaction provided between direct the proof of complementary thymine and adenine moieties, which resulted in an increased solubility of the block hydrogencopolymers bonding interactionby an improved between shielding nucleobases. of the hydrophobic blocks.

NH O 2 N NH N Base= N N O N

O 1) CuCl, bpy O O Br DMSO-d6, 30℃ O + O O 45 n O O 2) OTMS 45 O O O Base Polymers 2017, 9, 666 Base 4 of 25

SchemeScheme 1. Synthesis1. Synthesis of of copolymer copolymer poly(ethylenepoly(ethylene glycol) glycol) (PEG)–poly(nucleobase) (PEG)–poly(nucleobase) by ATRP by ATRP and and schematicschematic structure structure of assembled of assembled copolymers copolymers [26 ].[26]. Reproduced Reproduced with with permission. permission. Copyright Copyright John John Wiley and Sons,Wiley 2006.and Sons, 2006. Polymers 2017, 9, 666 5 of 25 In 2007, the same group reported the synthesis and controlled polymerization of all four nucleobases’ (thymine, adenine, cytosine, and guanine) methacrylate monomers through ATRP method [25]. Technically, by alkylation of the nucleobases with 3-bromopropyl methacrylate, thymine-, adenine-, cytosine-, and guanine-functionalized methacrylate monomers were prepared and their ATRP kinetics were examined. Interestingly, they found the formation of guanine and cytosine monomer with copper(II), and these two specific polymerizations required more addition of catalyst and the use of PMDETA. From this study, all four unprotected nucleobase monomers could be polymerized in a controlled manner, but there is still space for improving the level of control in the synthesis of nucleobase-containing polymers through ATRP. Similarly, Cheng et al. [47] synthesized poly[1-(4-vinylbenzyl)thymine] (PVBT) through ATRP at a high temperature (showed in Scheme 2) and, furthermore, performed nucleobase recognition through adding presynthesized 9-hexadecyladenine (A-C16) in the presence of polythymine homopolymers. Specifically, due to the hydrogen bonding interactions between the thymine groups of PVBTScheme and 2. Syntheticthe adenine procedures group used of to A-C16,obtain PVBT the andbiocomplementary A-C16 and graphical PVBT/A-C16 representations hierarchical of the Scheme 2. Synthetic procedures used to obtain PVBT and A-C16 and graphical representations of the supramolecularlamellar structures complexes of the were PVBT/A-C16 formed incomplexes dilute DM in SObulk solution. state [47]. In Reproduced the bulk state, with these permission. complexes lamellar structures of the PVBT/A-C16 complexes in bulk state [47]. Reproduced with permission. self-assembleCopyright into John well-ordered Wiley and Sons, lamellar 2008. structures; these self-assembled structures can be tuned by Copyrightdifferent A-C16 John Wiley loadings. and Sons,In 2015, 2008. a water-soluble triblock polymer and a pentablock-polymer containing3.2. Synthesizing nucleobases Nucleobase were Polymers synthesized by RAFT by Haddleton and Garcia et al. [27]. They utilized ATRP to polymerize methacryloyl uridine and methacryloyl adenosine from bromoisobutyroyl poly(ethylene As popular as ATRP, the RAFT (reversible addition-fragmentation chain transfer) process, glycol) (PEG) macroinitiators. Furthermore, they investigated the secondary structures of triblock which was first introduced by the group of Rizzardo [48] in 1998, is also recognized as one of the polymers and pentablock polymers in water by CD spectrum and mixing adenosine and uridine most versatile methods for producing a wide range of macromolecules with well-defined triblock copolymers in water resulted in a new supermolecular self-assembled structures, which functionalities and complex architectures. provided direct proof of hydrogen bonding interaction between nucleobases. In 2013, Kang et al. synthesized methacryloyl-monomers containing adenine and thymine to yield homopolymers and copolymers with excellent control over molecular weight and end group fidelity via RAFT polymerization (shown in Figure 2) [49]. Their results indicated that the polymerization solvent greatly affected the monomer reactivity; for example, chloroform promoted hydrogen bonds between the adenine monomers and thymine monomers, preferring moderate alternating copolymers, while solvent DMF suppressed these interactions, preferring statistical copolymers. Furthermore, they investigated the self-assembly properties of the copolymers and their TEM and DLS results clearly indicated that the presence or absence of hydrogen bonding between nucleobases could influence the morphology of forming micelles. Similarly, the same group explored more nucleobase-containing materials via RAFT dispersion polymerization in 1,4-dioxane and chloroform, and also investigated the behavior of copolymers self-assembly [29]. The results confirmed again that the morphologies of self-assembled nucleobase-containing polymers were dependent on polymerization solvents. For example: spherical micelles with internal structures were only observed in chloroform by varying the DP of the core-forming block. As an extension of this work, in 2015 they reported the synthesis and self-assembly of thymine-containing polymers via RAFT dispersion polymerization and a wide variety of morphologies were accessed by changing the different solvent of polymerization or incorporating adenine-containing mediators [50]. These results strongly demonstrated that nucleobase interactions played a central role in controlling the shape and size of polymeric self-assemblies.

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3.2. Synthesizing Nucleobase Polymers by RAFT As popular as ATRP, the RAFT (reversible addition-fragmentation chain transfer) process, which was first introduced by the group of Rizzardo [48] in 1998, is also recognized as one of the most versatile methods for producing a wide range of macromolecules with well-defined functionalities and complex architectures. In 2013, Kang et al. synthesized methacryloyl-monomers containing adenine and thymine to yield homopolymers and copolymers with excellent control over molecular weight and end group fidelity via RAFT polymerization (shown in Figure2)[ 49]. Their results indicated that the polymerization solvent greatly affected the monomer reactivity; for example, chloroform promoted hydrogen bonds between the adenine monomers and thymine monomers, preferring moderate alternating copolymers, while solvent DMF suppressed these interactions, preferring statistical copolymers. Furthermore, they investigated the self-assembly properties of the copolymers and their TEM and DLS results clearly indicated that the presence or absence of hydrogen bonding between nucleobases could influence the morphology of forming micelles. Similarly, the same group explored more nucleobase-containing materials via RAFT dispersion polymerization in 1,4-dioxane and chloroform, and also investigated the behavior of copolymers self-assembly [29]. The results confirmed again that the morphologies of self-assembled nucleobase-containing polymers were dependent on polymerization solvents. For example: spherical micelles with internal structures were only observed in chloroform by varying the DP of the core-forming block. As an extension of this work, in 2015 they reported the synthesis and self-assembly of thymine-containing polymers via RAFT dispersion polymerization and a wide variety of morphologies were accessed by changing the different solvent of polymerization or incorporating adenine-containing mediators [50]. These results strongly demonstrated that nucleobase interactions played a central role in controlling the shape and size of polymericPolymers 2017 self-assemblies., 9, 666 6 of 25

FigureFigure 2.2. SynthesisSynthesis andand self-assemblyself-assembly of adenineadenine and thyminethymine block copolymers using RAFT [[49].49]. ReproducedReproduced withwith permission.permission. CopyrightCopyright AmericanAmerican ChemicalChemical Society,Society, 2013. 2013.

Zhang et al. used a similar RAFT polymerization strategy to synthesize well-defined acrylic ABA triblock copolymers with nucleobase-functionalized external blocks and poly(n-butyl acrylate) (PnBA) as a central block (shown in Scheme 3) [31]. Moreover, they prepared triblock copolymer films and supramolecular blend and their results indicated that the acrylic monomer with a flexible spacer to the pendant nucleobases promoted intermolecular recognition of nucleobases, lowering the Tg of functionalized polymers. In addition, the complementary hydrogen bonding between adenine and thymine formed a thermally labile, physically crosslinked network that exhibited enhanced mechanical performance with melt processability, and the supramolecular blend exhibited a cylindrical microphase-separate morphology. More and more work has been using RAFT as an efficient and robust method for the preparation of multi-block polymers with nucleobase functionalities, and more and more morphologies have been accessed by fine tuning of the interactions between nucleobases [30,32,51]. RAFT has been the most frequently used living polymerization method in preparing nucleobase-containing polymers; however, it is still worth noting some challenges of this method. For example, RAFT usually requires careful selection of chain transfer agents (CTAs) for each monomer, which may require screening of CTAs before the polymerization of different nucleobase-containing monomers.

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Zhang et al. used a similar RAFT polymerization strategy to synthesize well-defined acrylic ABA triblock copolymers with nucleobase-functionalized external blocks and poly(n-butyl acrylate) (PnBA) as a central block (shown in Scheme3)[ 31]. Moreover, they prepared triblock copolymer films and supramolecular blend and their results indicated that the acrylic monomer with a flexible spacer to the pendant nucleobases promoted intermolecular recognition of nucleobases, lowering the Tg of functionalized polymers. In addition, the complementary hydrogen bonding between adenine and thymine formed a thermally labile, physically crosslinked network that exhibited enhanced mechanical performance with melt processability, and the supramolecular blend exhibited a cylindrical microphase-separate morphology. More and more work has been using RAFT as an efficient and robust method for the preparation of multi-block polymers with nucleobase functionalities, and more and more morphologies have been accessed by fine tuning of the interactions between nucleobases [30,32,51]. RAFT has been the most frequently used living polymerization method in preparing nucleobase-containing polymers; however, it is still worth noting some challenges of this method. For example, RAFT usually requires careful selection of chain transfer agents (CTAs) for each monomer, which may require screening of CTAs before the polymerization of different Polymersnucleobase-containing 2017, 9, 666 monomers. 7 of 25

Scheme 3. 3. SynthesisSynthesis of ofadenine- adenine- and and thymine-functiona thymine-functionalizedlized acrylic acrylic ABA ABAtriblock triblock copolymers copolymers using RAFTusing RAFTpolymerization polymerization and andschemati schematicc representation representation of ofsupramol supramolecularecular blend blend of of adenine adenine and thymine-functionalized triblock copolymers [[31].31]. Reproduced with permission.permission. Copyright Royal Society of Chemistry, 2015.2015.

3.3. Synthesizing Nucleobase Polymers by NMP As we we described described before, before, the the coordination coordination of nu ofcleobase nucleobase (guanine (guanine and cyotsine) and cyotsine) with a with metal a catalystmetal catalyst (e.g., CuBr) (e.g., usually CuBr) usuallyattenuates attenuates the polymerization the polymerization kinetics of kinetics ATRP. ofAs ATRP.such, nitroxide- As such, mediatednitroxide-mediated polymerization polymerization (NMP) could (NMP) be utilized could as be another utilized controlled as another radical controlled polymerization radical polymerization(CRP) technique (CRP) to produce technique nucleobase-containing to produce nucleobase-containing polymers [52,53]. polymers Mather et [52 al.,53 utilized]. Mather a novel et al. difunctionalutilized a novel alkoxyamine difunctional initiator alkoxyamine to polymerize initiator styrenic to polymerize nucleobase styrenic monomers nucleobase with poly( monomersn-butyl withacrylate) poly( homopolymersn-butyl acrylate) via homopolymersa two-step NMP via route a two-step [33] (Figure NMP 3). route The resulting [33] (Figure triblock3). The copolymers resulting containtriblock either copolymers adenine contain or thymine either as adenine outer blocks, or thymine and n as-butyl outer acrylate blocks, rubber and n-butyl as inner acrylate blocks. rubber Once adenineas inner blocks.and thymine Once adeninepolymers and are thymine mixed polymersin chloroform, are mixed the inviscosity chloroform, of the the blend viscosity increased of the dramatically,blend increased indicating dramatically, the existence indicating of thehydrog existenceen-bonding of hydrogen-bonding interactions between interactions complementary between nucleobase subunits since viscosity is a function of effective molecular weight. Furthermore, the annealed mixture in the solid state revealed a higher softening temperature and presented further proof of a complementary A–T hard phase. Although the NMP method avoids using a metal catalyst in polymerization, it still has some inherent challenges that limit its wide application in preparing nucleobase-containing polymers. For example, the slow polymerization kinetics usually requires high temperatures and a long polymerization time.

Figure 3. Synthesis of adenine and thymine nucleobase-functionalized triblock copolymers using NMP [33]. Reproduced with permission. Copyright American Chemical Society, 2007.

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Scheme 3. Synthesis of adenine- and thymine-functionalized acrylic ABA triblock copolymers using RAFT polymerization and schematic representation of supramolecular blend of adenine and thymine-functionalized triblock copolymers [31]. Reproduced with permission. Copyright Royal Society of Chemistry, 2015.

3.3. Synthesizing Nucleobase Polymers by NMP As we described before, the coordination of nucleobase (guanine and cyotsine) with a metal catalyst (e.g., CuBr) usually attenuates the polymerization kinetics of ATRP. As such, nitroxide- mediated polymerization (NMP) could be utilized as another controlled radical polymerization (CRP) technique to produce nucleobase-containing polymers [52,53]. Mather et al. utilized a novel difunctional alkoxyamine initiator to polymerize styrenic nucleobase monomers with poly(n-butyl acrylate) homopolymers via a two-step NMP route [33] (Figure 3). The resulting triblock copolymers contain either adenine or thymine as outer blocks, and n-butyl acrylate rubber as inner blocks. Once Polymers 2017, 9, 666 7 of 24 adenine and thymine polymers are mixed in chloroform, the viscosity of the blend increased dramatically, indicating the existence of hydrogen-bonding interactions between complementary nucleobasecomplementary subunits nucleobase since viscosity subunits is sincea function viscosity of effective is a function molecular of effective weight. molecularFurthermore, weight. the annealedFurthermore, mixture the annealedin the solid mixture state re invealed the solid a higher state softening revealed atemperature higher softening and presented temperature further and presentedproof of a complementary further proof of aA–T complementary hard phase. Although A–T hard phase.the NMP Although method the avoids NMP using method a metal avoids catalyst using ina metal polymerization, catalyst in polymerization,it still has some it inherent still has somechalle inherentnges that challenges limit its wide that limitapplication its wide in application preparing nucleobase-containingin preparing nucleobase-containing polymers. For polymers. example, For the example, slow polymerization the slow polymerization kinetics usually kinetics requires usually highrequires temperatures high temperatures and a long and polymerization a long polymerization time. time.

Figure 3. Synthesis of adenine and thymine nucleobase-functionalizednucleobase-functionalized triblock copolymers using NMP [33]. [33]. Reproduced with permission. Co Copyrightpyright American Chemical Society, 2007.2007.

3.4. Synthesizing Nucleobase Polymers by Conventional Radical Polymerization Besides controlled polymerizations, conventional radical polymerization also allows efficient large-scale synthesis of nucleobase functionalized polymers. As early as 2005, Luts et al. investigated the polymerization of a series of monomers bearing nucleobases 1-(4-vinylbenzyl)uracil (VBU), 1-(4-vinylbenzyl)thymine (VBT), and 9-(4-vinylbenzyl)adenine (VBA) [37]. The corresponding homopolymers (PVBT, PVBU, and PVBA) were prepared in high yields via conventional radical polymerization, while the copolymers of dodecyl methacrylate (DMA) with either VBT or VBA were prepared by ATRP and free radical polymerization. The results indicated that these homopolymers were not very practical due to poor solubility in most of the common organic solvents. However, copolymers of DMA with either VBT or VBA could be dissolved in a range of organic solvents. Furthermore, the formed copolymers P(VBT-co-DMA) and P(VBA-co-DMA) were found to self-assemble in dilute solutions in dioxane or chloroform, as confirmed by the hypochromicity effect in UV spectroscopy. Nevertheless, it was found that at higher concentrations of chloroform, P(VBT-co-DMA), P(VBA-co-DMA), or P(VBT-co-DMA)/P(VBA-co-DMA) mixtures spontaneously self-assembled into spherical aggregates. Later in 2006, the same group studied in detail the self-organization of random copolymers composed of a nucleobase monomer (either VBT or VBA) and DMA in dilute chloroform [35]. They investigated how the balance between the molar fractions of the nucleobase monomer and DMA could affect the solubility and self-assembly behavior of nucleobase-containing copolymers. In addition, they demonstrated that hydrogen bonding was an efficient driving force for organizing synthetic nucleobase-containing polymers in a noncompetitive solvent. Nakahata presented an interesting work that created a selective macroscopic self-assembly system by using polymer gels modified with either complementary DNA oligonucleotides or nucleobase-containing polymers [36]. Specifically, hydrogels and organogels were obtained via conventional radical terpolymerization of AAm or styrene (main chain units) and MBAAm or DVB (crosslinking units) (showed in Scheme4). The oligonucleotide-modified hydrogels exhibited macroscopic recognition between complementary strands. Similarly, nucleobase-modified organogels exhibited the same adhesion as DNA oligonucleotide-functionalized hydrogels and were visualized on a macroscopic scale. Direct adhesion between macroscopic objects by nucleobase interaction provides the possibility of creating “smart” materials that can bind many kinds of heterogeneous materials Polymers 2017, 9, 666 8 of 25

3.4. Synthesizing Nucleobase Polymers by Conventional Radical Polymerization Besides controlled polymerizations, conventional radical polymerization also allows efficient large-scale synthesis of nucleobase functionalized polymers. As early as 2005, Luts et al. investigated the polymerization of a series of monomers bearing nucleobases 1-(4-vinylbenzyl)uracil (VBU), 1-(4- vinylbenzyl)thymine (VBT), and 9-(4-vinylbenzyl)adenine (VBA) [37]. The corresponding homopolymers (PVBT, PVBU, and PVBA) were prepared in high yields via conventional radical polymerization, while the copolymers of dodecyl methacrylate (DMA) with either VBT or VBA were prepared by ATRP and free radical polymerization. The results indicated that these homopolymers were not very practical due to poor solubility in most of the common organic solvents. However, copolymers of DMA with either VBT or VBA could be dissolved in a range of organic solvents. Furthermore, the formed copolymers P(VBT-co-DMA) and P(VBA-co-DMA) were found to self- assemble in dilute solutions in dioxane or chloroform, as confirmed by the hypochromicity effect in UV spectroscopy. Nevertheless, it was found that at higher concentrations of chloroform, P(VBT-co- DMA), P(VBA-co-DMA), or P(VBT-co-DMA)/P(VBA-co-DMA) mixtures spontaneously self- assembled into spherical aggregates. Later in 2006, the same group studied in detail the self- organization of random copolymers composed of a nucleobase monomer (either VBT or VBA) and DMA in dilute chloroform [35]. They investigated how the balance between the molar fractions of the nucleobase monomer and DMA could affect the solubility and self-assembly behavior of nucleobase-containing copolymers. In addition, they demonstrated that hydrogen bonding was an efficient driving force for organizing synthetic nucleobase-containing polymers in a noncompetitive solvent. Nakahata presented an interesting work that created a selective macroscopic self-assembly system by using polymer gels modified with either complementary DNA oligonucleotides or nucleobase-containing polymers [36]. Specifically, hydrogels and organogels were obtained via conventional radical terpolymerization of AAm or styrene (main chain units) and MBAAm or DVB (crosslinking units) (showed in Scheme 4). The oligonucleotide-modified hydrogels exhibited macroscopic recognition between complementary strands. Similarly, nucleobase-modified organogels exhibited the same adhesion as DNA oligonucleotide-functionalized hydrogels and were visualized on a macroscopic scale. Direct adhesion between macroscopic objects by nucleobase Polymers 2017, 9, 666 8 of 24 interaction provides the possibility of creating “smart” materials that can bind many kinds of heterogeneous materials without adhesives. Although conventional radical polymerization provides withouta very simple adhesives. method Although for nucleobase-containing conventional radical polymer polymerization synthesis provides (no metal a catalyst, very simple no precursors), method for nucleobase-containingit also has disadvantages polymer such synthesisas insufficient (no metal control catalyst, of the no polymer precursors), length, it also polydispersity, has disadvantages and suchmacromolecular as insufﬁcient structure, control which of the may polymer impair length, its potential polydispersity, applications and macromolecular in biology and structure,materials whichscience. may impair its potential applications in biology and materials science.

Scheme 4. Chemical structures of the nucleobase organogels and a schematic illustration of their macroscopic self-assembly [[36].36]. Reproduced with permission.permission. Copyright John Wiley andand Sons,Sons, 2015.2015.

With the inherent propertyproperty ofof sequencesequence recognition,recognition, McHale et al. reported that the nucleobase core of a blockblock copolymercopolymer micelle can serve as a templatetemplate for controlledcontrolled radical polymerization [[28]28] (Figure 4). This uniquely templated polymerization yields unexpectedly high molecular weight (FigurePolymers 20174)., This9, 666 uniquely templated polymerization yields unexpectedly high molecular weight9 of 25 polymers (up(up toto ~400,000~400,000 g/mol),g/mol), with with low polydispersity (P ≤ 1.08);1.08); and and their their TEM imaging clearly demonstrated that polymerization takes place in the core of the micelle as its size changes from ~13 to demonstrated that polymerization takes place in the core of the micelle as its size changes from ~13 ~20to ~20 nm nm before before and and after after polymerization. polymerization. More More interestingly, interestingly, the discrete the discrete micelle micelle core structures core structures enabled enabledthe control the of control the polymerization of the polymerization by segregation by segregation of propagating of propagating radicals. radicals.

(a)

(b)

:PSt-b-PVBT

Figure 4. ((aa)) Self-assembly Self-assembly of of nucleobase nucleobase polymer polymer templates templates into into micelles and ( b) the templatedtemplated polymerization ofof complementary complementary nucleobase nucleobase monomers monomers [28]. Reproduced [28]. Reproduced with permission. with permission. Copyright CopyrightSpringer Nature, Springer 2012. Nature, 2012.

3.5. Synthesizing Nucleobase Polymers by Click Chemistry “Click chemistry” is a concept introduced by Sh Sharplessarpless [54] [54] in 2001 and since then has become one of the the most most powerful powerful paradigms paradigms in in polymer polymer chemistry chemistry and and materials materials science science [55–57]. [55–57 Due]. Due to its to simplicityits simplicity and and efficiency, efﬁciency, click click chemistry chemistry has has been been extensively extensively employed employed for for accessing linear polymers [58–63], branched polymers [64–73], dendrimers [74–79], and polymer networks [80–85]. The combination of click chemistry and nucleobase-containing polymers is promising. For example, in 2014 Han et al. reported a facile and scalable method to synthesize DNA-mimetic sequential polymers with tailored hetero-functional groups based on radical-initiated thiol-yne click polymerization [39]. Firstly, radicals were generated from either a UV or thermal initiator, yielding a thiyl radical, enabled stepwise propagation, and polymer chain transfer (shown in Figure 5). With a delicate selection of monomers with different functionalities, various types of sequenced polymers such as linear sequence, periodic sequence, random sequence, and hyperbranched sequence were obtained (Figure 5). Furthermore, to achieve single-strand DNA-mimetic macromolecules, alternating adenine and thymine groups were introduced by another classic click chemistry: Cu- catalyzed azide and alkyne cycloaddition (CuAAC). This work is an excellent example of utilizing two orthogonal click reactions to synthesize nucleobase-containing polymers from simple starting substrates in two simple steps, indicating the power of click chemistry. In 2015, Xi et al. [38] synthesized nucleobase-containing sequence-controlled polymers (homopolymers of a single nucleobase or homopolymers of specific repeating nucleobase sequences) by combining orthogonal thiol-ene and thiol-Michael click chemistry reactions (Scheme 5) [86–89]. In this case, a thiol-protected acrylamide monomer is coupled to a functionalized thiol–allylamine monomer in a thiol-Michael addition, then the thiol group is deprotected to restore the thiol and polymerized with allyamine functional groups by radical initiation of the thiol-ene reaction. This “click” by “click” approach is a great demonstration of synthesizing nucleobase-functionalized sequence-controlled periodic copolymers/oligomers and polymers without the use of a solid support. In addition, these nucleobase-containing polymers were employed in various applications in

Polymers 2017, 9, 666 9 of 24 Polymers 2017, 9, 666 10 of 25 polymersmaterials science, [58–63], such branched as sequence-controlled polymers [64–73], dendrimersassembly of [nanoparticles,74–79], and polymer DNA delivery networks [90], [80 –and85]. Theorganogel combination formation of click [91]. chemistry and nucleobase-containing polymers is promising. For example, in 2014 Han et al. reported a facile and scalable method to synthesize DNA-mimetic sequential polymers with tailored hetero-functional groups based on radical-initiated thiol-yne click polymerization [39]. Firstly, radicals were generated from either a UV or thermal initiator, yielding a thiyl radical, enabled stepwise propagation, and polymer chain transfer (shown in Figure5). With a delicate selection of monomers with different functionalities, various types of sequenced polymers such as linear sequence, periodic sequence, random sequence, and hyperbranched sequence were obtained (Figure5). Furthermore, to achieve single-strand DNA-mimetic macromolecules, alternating adenine and thymine groups were introduced by another classic click chemistry: Cu-catalyzed azide and alkyne cycloaddition (CuAAC). This work is an excellent example of utilizing two orthogonal click reactions to synthesize nucleobase-containing polymers from simple starting substrates in two simple steps, indicating the power of click chemistry. In 2015, Xi et al. [38] synthesized nucleobase-containing sequence-controlled polymers (homopolymers of a single nucleobase or homopolymers of speciﬁc repeating nucleobase sequences) by combining orthogonal thiol-ene and thiol-Michael click chemistry reactions (Scheme5)[ 86–89]. In this case, a thiol-protected acrylamide monomer is coupled to a functionalized thiol–allylamine monomer in a thiol-Michael addition, then the thiol group is deprotected to restore the thiol and polymerized with allyamine functional groups by radical initiation of the thiol-ene reaction. This “click” by “click” approachFigure is 5.a Sequence-controlled great demonstration hetero-functional, of synthesizing topological nucleobase-functionalized polymers (R1 and R2 denote sequence-controlled two different periodicfunctional copolymers/oligomers groups and S represents and a polymersthioether bo withoutnd, which the can use be transfor of a solidmed support.into a sulfone In bond addition, theseby nucleobase-containing oxidation) and the syntheti polymersc route were to employed a DNA-mimetic in various nucleo applicationsbase-containing in materials polymer science, [39]. such as sequence-controlledReproduced with permission. assembly Co ofpyright nanoparticles, Springer DNANature, delivery 2014. [90], and organogel formation [91].

Scheme 5.5.( A(A) General) General monomer monomer structure structure for thiol-based for thiol-based click polymerization click polymerization (R represents (R represents functional group,functional including group, but including not limited but tonot nucleobases); limited to nucleobases); (B) The two types (B) The of thioltwo clicktypes reaction; of thiol (clickC) Thiol-click reaction. reaction(C) Thiol-click strategy reaction for sequence-controlled strategy for sequence-controlled periodic functional periodic oligomers functional and polymers oligomers [38 ].and Reproduced polymers with[38]. Reproduced permission. Copyrightwith permission. John Wiley Copyright and Sons, John 2015. Wiley and Sons, 2015.

Polymers 2017, 9, 666 10 of 25 materialsPolymers 2017 science,, 9, 666 such as sequence-controlled assembly of nanoparticles, DNA delivery [90],10 and of 24 organogel formation [91].

1 2 Figure 5. Sequence-controlledSequence-controlled hetero-functional, topologicaltopological polymerspolymers (R(R1 and R2 denotedenote two two different different functional groups groups and and S Srepresents represents a thioether a thioether bond, bond, which which can canbe transfor be transformedmed into a into sulfone a sulfone bond bybond oxidation) by oxidation) and andthe thesyntheti syntheticc route route to toa aDNA-mimetic DNA-mimetic nucleo nucleobase-containingbase-containing polymer polymer [39]. [39]. Reproduced with permission. Co Copyrightpyright Springer Nature, 2014.

In 2017, Liu et al. [40] presented a synthesis of an intrinsically water-soluble single-stranded DNA analog formed by the synthesis of a Clickable Nucleic Acid (CNA). They formed molecules with pendantPolymers 2017 hydroxyl, 9, 666 groups present on the main polymer backbone, and subsequent modiﬁcation of11 those of 25 hydroxyls with sulfonate moieties further enhances the hydrophilicity (Figure6). The CNA polymer withpolymer one with pendant one hydroxylpendant hydroxyl group for group each dimerfor each was dimer obtained was byobtained the radical-mediated by the radical-mediated thiol-ene polymerizationthiol-ene polymerization approach. Aapproach. few simple A steps few weresimple needed steps to were functionalize needed theto freefunctionalize hydroxyl groupthe free on thehydroxyl polymer group backbone on the topolymer obtain thebackbone water-soluble to obtain CNA the water-soluble polymer. Furthermore, CNA polymer. they demonstrated Furthermore, thatthey this demonstrated water-soluble that CNA this polymerwater-soluble was capable CNA polymer of binding was to complementarycapable of binding DNA to oncomplementary the surface of nanoparticles.DNA on the surface Although of nanoparticles. click chemistry Although enables fastclick and chemistry efﬁcient enables preparation fast and of nucleobase-containing efficient preparation polymers,of nucleobase-containing there is still a lot polymers, of space there for researchers is still a lot to of optimize space for synthetic researchers strategies/procedures to optimize synthetic to achievestrategies/procedures longer polymers to achieve and sequence longercontrol polymers in theand future. sequence control in the future.

Scheme 5. (A) General monomer structure for thiol-based click polymerization (R represents functional group, including but not limited to nucleobases); (B) The two types of thiol click reaction. (C) Thiol-click reaction strategy for sequence-controlled periodic functional oligomers and polymers [38]. Reproduced with permission. Copyright John Wiley and Sons, 2015. Figure 6.6. Schematic illustration of water-solublewater-soluble CNACNA polymerpolymer synthesissynthesis [[40].40]. ReproducedReproduced withwith Inpermission.permission. 2017, Liu Copyright et al. [40] Royal presented SocietySociety ofofa Chemistry,Chemistry,synthesis 2017.of2017. an intrinsically water-soluble single-stranded DNA analog formed by the synthesis of a Clickable Nucleic Acid (CNA). They formed molecules with3.6. Synthesizing pendant hydroxyl Nucleobase groups Polymers present by on ROMP the main polymer backbone, and subsequent modification of thoseAs ahydroxyls a variation variation ofwith ofthe sulfonate theolefin oleﬁn metathesis moieties metathesis reaction further reaction, ,enhances ring-opening ring-opening the hy metathesisdrophilicity metathesis polymerization (Figure polymerization 6). The (ROMP) CNA (ROMP) [92,93] represents [92,93] represents another widely another used widely method used to synthesize method to polymer synthesize materials polymer with materials tunable sizes, with shapes, and functions, especially in the preparation of nucleobase-containing polymers. Bazzi et al. [94] first reported the preparation of “DNA-mimetic” homopolymers and block copolymers through controlled ring-opening metathesis polymerization of non-covalently protected monomers as early as 2002. Notably, an adenine-containing monomer was “protected” by succinimide, based on the hydrogen bonding between succinimide and the adenine moiety. Additionally, this simple introduction of succinimide increased the solubility of the resulting polymer, which prevents resultant polymers from precipitating before the reaction is complete. These nucleobase-containing block copolymers were produced by following the same ROMP procedure. Finally, the self-assembly of copolymers (m = 50, n = 5; shown in red in Figure 7) was investigated in THF solution and the results revealed the ability of these hydrogen-bonding-enabled self-complementary block copolymers to self-assemble into novel nanoscale rod morphologies.

Polymers 2017, 9, 666 11 of 24 tunable sizes, shapes, and functions, especially in the preparation of nucleobase-containing polymers. Bazzi et al. [94] ﬁrst reported the preparation of “DNA-mimetic” homopolymers and block copolymers through controlled ring-opening metathesis polymerization of non-covalently protected monomers as early as 2002. Notably, an adenine-containing monomer was “protected” by succinimide, based on the hydrogen bonding between succinimide and the adenine moiety. Additionally, this simple introduction of succinimide increased the solubility of the resulting polymer, which prevents resultant polymers from precipitating before the reaction is complete. These nucleobase-containing block copolymers were produced by following the same ROMP procedure. Finally, the self-assembly of copolymers (m = 50, n = 5; shown in red in Figure7) was investigated in THF solution and the results revealed the ability of these hydrogen-bonding-enabled self-complementary block copolymers to self-assemble into novelPolymers nanoscale 2017, 9 rod, 666 morphologies. 12 of 25

FigureFigure 7. (A 7.) ROMP (A) ROMP with with noncovalently noncovalently protected protected monomer;monomer; ( (BB) )preparation preparation of ofblock block copolymers copolymers via via ROMPROMP [94]. [94]. Reproduced Reproduced with with permission. permission. Copyright Copyright American Chem Chemicalical Society, Society, 2006. 2006.

After that, a similar ROMP strategy was utilized by Lo et al. [41] in the construction of a thymine- Aftercontaining that, block a similar copolymer ROMP with strategy narrow wasmolecular utilized weight by Lodistribution. et al. [41 As] in shown the construction in Scheme 6, of a thymine-containingadenine-containing block monomers copolymer were withaligned narrow with hydrogen-bonding molecular weight interactions distribution. to the As thymine shown in Schemetemplate,6, adenine-containing and subsequently monomers underwent were Sonogashira aligned withpolymerization, hydrogen-bonding leading to interactions the templated to the thyminesynthesis template, of a andconjugated subsequently polyme underwentr. The daughter Sonogashira strand is polymerization,found to possess leading a narrow to themolecular templated synthesisweight of distribution a conjugated and polymer. a chain length The nearly daughter equiva strandlent to is that found of the to parent possess template, a narrow indicating molecular weightthat distribution the daughter and strand a chain could lengthcopy the nearly length and equivalent distribution to that information of the parent from the template, template strand. indicating However, non-templated polymerization or polymerization with the incorrect template generated a that the daughter strand could copy the length and distribution information from the template strand. short conjugated oligomer with a significantly broader molecular weight distribution. However, non-templated polymerization or polymerization with the incorrect template generated a short conjugated oligomer with a significantly broader molecular weight distribution. Peptide nucleic acids (PNAs) are synthetic DNA mimetics whose backbones are electrical neutral amide bonds, which contribute high stability towards nuclease degradation and high affinity towards complementary DNA/RNA strands [95–98]. James et al. [42] reported a preparation of polymeric nucleic acids wherein single-stranded sequences of PNAs are incorporated as polymer brushes via graft-through polymerization using the ROMP initiator “Ru”. This work clearly demonstrated the power of ROMP in preparing amphiphilic brush copolymers by graft-through polymerization of a relatively large side chain: oligonucleotides (Figure8). This approach of combining PNAs and ROMP could provide an efficient strategy for the incorporation of nucleic acids into particles and polymer-based materials, which might be useful for various applications such as affinity purification

Scheme 6. Templated polymerization of nucleobase-containing monomers [41]. Reproduced with permission. Copyright American Chemical Society, 2009.

Polymers 2017, 9, 666 12 of 25

Figure 7. (A) ROMP with noncovalently protected monomer; (B) preparation of block copolymers via ROMP [94]. Reproduced with permission. Copyright American Chemical Society, 2006.

After that, a similar ROMP strategy was utilized by Lo et al. [41] in the construction of a thymine- Polymers 2017, 9, 666 12 of 24 containing block copolymer with narrow molecular weight distribution. As shown in Scheme 6, adenine-containing monomers were aligned with hydrogen-bonding interactions to the thymine of DNA,template, gene and delivery, subsequently and programmable underwent Sonogashira materials. Likepolymerization, other polymerization leading to the strategies, templated ROMP synthesis of a conjugated polymer. The daughter strand is found to possess a narrow molecular provides another efﬁcient method to access nucleobase-containing polymers; however, the driving weight distribution and a chain length nearly equivalent to that of the parent template, indicating forcethat of ROMP the daughter is the strand relief could of ring copy strain the length in cyclic and monomers,distribution information which usually from requiresthe template the strand. additional step ofHowever, preparing non-templated nucleobase-functionalized polymerization or cyclicpolymeri oleﬁnszation before with the polymerization. incorrect template The generated cost of thea Ru catalystshort is conjugated another concern, oligomer especially with a significan in large-scaletly broader polymer molecular preparations. weight distribution.

Polymers 2017, 9, 666 13 of 25

Peptide nucleic acids (PNAs) are synthetic DNA mimetics whose backbones are electrical neutral amide bonds, which contribute high stability towards nuclease degradation and high affinity towards complementary DNA/RNA strands [95–98]. James et al. [42] reported a preparation of polymeric nucleic acids wherein single-stranded sequences of PNAs are incorporated as polymer brushes via graft-through polymerization using the ROMP initiator “Ru”. This work clearly demonstrated the power of ROMP in preparing amphiphilic brush copolymers by graft-through polymerization of a relatively large side chain: oligonucleotides (Figure 8). This approach of combining PNAs and ROMP could provide an efficient strategy for the incorporation of nucleic acids into particles and polymer-based materials, which might be useful for various applications such as affinity purification of DNA, gene delivery, and programmable materials. Like other polymerization strategies, ROMP provides another efficient method to access nucleobase-containing polymers; however, the driving force of ROMP is the relief of ring strain in cyclic monomers, which usually requires the additional step of preparing nucleobase-functionalized cyclic olefins before polymerization.SchemeScheme 6. Templated 6. Templated The cost polymerization polymerizationof the Ru catalyst of of nucleobase-containing nucleobase-con is another concern,taining monomers monomersespecially [41]. in [41 Reproducedlarge-scale]. Reproduced withpolymer with permission. Copyright American Chemical Society, 2009. permission.preparations. Copyright American Chemical Society, 2009.

FigureFigure 8. Preparation 8. Preparation of poly(oligonucleotide):of poly(oligonucleotide): PNA homopolymer homopolymer (A ();A Poly-PNA); Poly-PNA block block copolymer copolymer (B)[42(B].) [42]. Reproduced Reproduced with with permission. permission. Copyright Copyright American American Chemical Chemical Society, Society, 2014. 2014.

Most recently, Gregory et al. [99] presented a novel work that using a ring-opening polymerizationMost recently, Gregorymethod etother al. [ 99than] presented ROMP. aIn novel this work work, that thymidine-based using a ring-opening cyclic polymerizationcarbonate methodincorporating other than CO ROMP.2 at 1 Inatm this pressure work, thymidine-based and this monomer cyclic underwent carbonate ROP incorporating under mild COreaction2 at 1 atm pressureconditions and this with monomer organocatalyst underwent TBD and ROP 4-MeBnOH under mild alcohol reaction initiator conditions to yield novel with thymidine-based organocatalyst TBD and 4-MeBnOHpolycarbonates. alcohol Similarly, initiator Tsao to yield[43] novelet al. thymidine-basedpresented a synthetic polycarbonates. strategy that Similarly, employed Tsao [43] organocatalytic ROP of a six-membered cyclic phosphoester monomer at ambient temperature to afford a new type of thymidine-derived 3′,5′-linked polyphosphoester with a butenyl group located on each repeat unit. This is the first time such well-defined DNA-derived polyphosphoesters with 3′,5′-linkages in the backbones have been prepared via ROP.

Polymers 2017, 9, 666 13 of 24 et al. presented a synthetic strategy that employed organocatalytic ROP of a six-membered cyclic phosphoester monomer at ambient temperature to afford a new type of thymidine-derived 30,50-linked polyphosphoester with a butenyl group located on each repeat unit. This is the ﬁrst time such well-deﬁned DNA-derived polyphosphoesters with 30,50-linkages in the backbones have been prepared Polymers 2017, 9, 666 14 of 25 via ROP.

3.7. Synthesizing Nucleobase Po Polymerslymers by Other Polymerizations ̈ The nucleobasenucleobase chemistrychemistry has has also also been been overlapped overlapped with with conducting conducting polymers. polymers. Bäuerle Bauerle et al. et [ 44al.] presented[44] presented the synthesis, the synthesis, characterization, characterization, and polymerization and polymerization of bithiophenes of bithiophenes with the nucleobases with the nucleobases uracil and adenine as sidechains, and found the molecular recognition could influence uracil and adenine as sidechains, and found the molecular recognition could inﬂuence the electronic the electronic properties of the polythiophenes, e.g., the presence of a complementary base could properties of the polythiophenes, e.g., the presence of a complementary base could induce an increase induce an increase in the oxidation potential for polymers (Shown in Figure 9). in the oxidation potential for polymers (Shown in Figure9).

Figure 9. Structures of nucleobase-functionalized polyth polythiophenesiophenes [44]. [44]. Reproduced Reproduced with with permission. permission. Copyright John Wiley and Sons, 1998.1998.

4. Applications of Nucleobase-Containing Polymers in Materials Science

4.1. Hydrogels Hydrogels have served as matrices for 3D cell culture with widespread applications in tissue engineering, biomolecule delivery, and regenerative medicine [[100,101].100,101]. Based on the crosslinkingcrosslinking mechanism, hydrogels can be clas classiﬁedsified into physically crosslinkedcrosslinked hydrogels and chemically crosslinked hydrogels. Although chemically crossl crosslinkedinked hydrogels usually provide a stable polymer network with slow slow degradation degradation kinetics, kinetics, physically physically crosslinked crosslinked hydrogels hydrogels have have also also attracted attracted a lot a lotof attention,of attention, especially especially as injectable as injectable biomaterials biomaterials in translational in translational medicine medicine [102]. Among [102]. Amongall the physical all the physicalcrosslinked crosslinked mechanisms mechanisms for hydrogel for hydrogel formation, formation, hydrogen hydrogen bonding bonding remains remains one of onethe ofmost the frequentlymost frequently used usedmethods, methods, which which makes makes nucleobase-containing nucleobase-containing polymers polymers an ideal an ideal candidate candidate for physicallyfor physically crosslinked crosslinked hydrogels. hydrogels. In 2012, In 2012, Tan Tanet al. et developed al. developed a biological a biological hydrogel hydrogel that self- that assembledself-assembled via vianucleobase nucleobase interaction interaction from from adenineadenine- and and thymine-functionalized thymine-functionalized tetra-arm tetra-arm PEG polymers (Scheme 77))[ [103].103]. This hydrogel exhibi exhibitedted both thermal and mech mechanicalanical responsiveness, such as as gel-sol gel-sol transition transition under under high high strain strain cond conditionsitions or orhigh high temperature, temperature, which which could could break break the physicalthe physical linkage linkage between between adenine adenine and and thymine. thymine. Additionally, Additionally, they they demonstrated demonstrated that that this nucleobase-interaction-based hydrogel can serve as an injectable scaffold for growth factor delivery and cell encapsulation, which indicated the excellent biocompatibility of these materials and their potential for use in further biomedical applications.

Polymers 2017, 9, 666 14 of 24 Polymers 2017, 9, 666 15 of 25

Polymers 2017, 9, 666 15 of 25

SchemeScheme 7. 7. Poly(ethylene glycol)-maleimide (PEG-Mal) was functionalized with (a) thiola thymine (T- Poly(ethylene glycol)-maleimide (PEG-Mal) was functionalized with ( ) thiol thymine (T-SH)SH) and and ( (bb) )thiol thiol adenine adenine (A-SH); (A-SH); (c) (Schematicc) Schematic of the of biological the biological self-assembly self-assembly of ideal of four-arm ideal four-arm PEG PEGhydrogelScheme hydrogel 7.network Poly(ethylene network via via intermolecular glycol)-maleimide intermolecular nucleoba (P nucleobaseEG-Mal)se interactionwas interaction functionalized between between with thymine (a thymine) thiol thymineand and adenine adenine(T- functionalitiesfunctionalitiesSH) and (b) [ 103thiol [103].]. adenine Reproduced Reproduced (A-SH); with with (c) Schematic permission. permission. of the CopyrightCopyright biological Ro Royal selyalf-assembly Society Society ofof of Chemistry,ideal Chemistry, four-arm 2012. 2012. PEG hydrogel network via intermolecular nucleobase interaction between thymine and adenine 4.2.4.2. DNA DNAfunctionalities Delivery Delivery [103]. Reproduced with permission. Copyright Royal Society of Chemistry, 2012. DNA delivery, especially non-viral DNA delivery, is a powerful and popular tool for gene DNA4.2. DNA delivery, Delivery especially non-viral DNA delivery, is a powerful and popular tool for gene silencing silencing in various diseases [104–107]. However, there are still challenges to building an efficient in various diseases [104–107]. However, there are still challenges to building an efficient delivery deliveryDNA system. delivery, In most especially cases, non-viralcationic polymersDNA delivery, have isbeen a powerful widely studiedand popular and investigatedtool for gene for system. In most cases, cationic polymers have been widely studied and investigated for DNA delivery, DNAsilencing delivery, in various but they diseases usually [104–107]. encounter However, some ditherefficulties are still such challenges as low DNAto building uptake, an slowefficient DNA but theydelivery usually system. encounter In most somecases, difficultiescationic polymers suchas have low been DNA widely uptake, studied slow and DNA investigated release, andfor lack release, and lack of nuclear targeting [108,109]. To expand the materials toolbox for DNA delivery, DNA delivery, but they usually encounter some difficulties such as low DNA uptake, slow DNA of nuclearHarguindey targeting et al. utilized [108,109 synthetic]. To expand DNA theanalogs materials (CNAs) toolbox and synthesized for DNA triblock delivery, copolymerHarguindey (PEG- et al. release, and lack of nuclear targeting [108,109]. To expand the materials toolbox for DNA delivery, utilizedCNA-PLGA) synthetic for DNA sequester analogs high (CNAs) loading and of synthesizedDNA [90]. These triblock synthetic copolymer triblock (PEG-CNA-PLGA) copolymers were for Harguindey et al. utilized synthetic DNA analogs (CNAs) and synthesized triblock copolymer (PEG- sequestertranslated high into loading polymer of DNA nanoparticles [90]. These through synthetic an triblock emulsion copolymers technique. were In addition, translated these into polymerCNA- nanoparticlesCNA-PLGA) through for sequester an emulsion high loading technique. of DNA In addition, [90]. These these synthetic CNA-containing triblock copolymers particles were exhibited containingtranslated particlesinto polymer exhibited nanoparticles high encapsul throughation an emulsionof complementary technique. InDNA addition, sequences these CNA-through high encapsulation of complementary DNA sequences through specific nucleobase recognition specificcontaining nucleobase particles recognition exhibited interactions,high encapsul whicationh demonstratedof complementary the potential DNA sequences application through of these interactions,CNA-containingspecific nucleobase which particles demonstrated recognition as DNA interactions, thecarriers potential for whic specific applicationh demonstrated gene silencing of thesethe potential(Figure CNA-containing 10). application of particles these as DNACNA-containing carriers for specific particles gene as silencingDNA carriers (Figure for specific 10). gene silencing (Figure 10).

Figure 10. DNA loading measurement of triblock PEG-CNA-PLGA nanoparticles [90]. Reproduced Figure 10. DNA loading measurement of triblock PEG-CNA-PLGA nanoparticles [90]. Reproduced Figurewith 10.permission.DNA loading Copyright measurement John Wiley of and triblock Sons, PEG-CNA-PLGA2017. nanoparticles [90]. Reproduced with permission. Copyright John Wiley and Sons, 2017. with permission. Copyright John Wiley and Sons, 2017.

Polymers 2017,, 9,, 666666 1516 of 2425

4.3. Self-Healing Materials 4.3. Self-Healing Materials Self-healing materials include but are not limited to metals, polymers, and composites that have the capabilitySelf-healing to heal materials and recover include the but original are not stat limitede after to damage metals, (e.g., polymers, mechanical, and composites thermal) [110,111]. that have theOver capability the last decade, to heal andthey recover have attracted the original much state attention after damage and been (e.g., investigated mechanical, intensely thermal) [112–115]. [110,111]. OverDepending the last on decade, the nature they of have the attractedself-healing much process, attention it can and be been classified investigated into two intensely types: autonomic [112–115]. Dependingand non-autonomic on the nature [110]. ofSpecific the self-healingally, non-autonomic process, it self-healing can be classified materials into twoneed types: external autonomic energy, andwhile non-autonomic autonomic materials [110]. Specifically, do not require non-autonomic extra stimulation self-healing for materials self-healing. need Complementary external energy, whilenucleobase-containing autonomic materials polymers do have not require a built-in extra advantage stimulation in the preparation for self-healing. of materials Complementary that exhibit nucleobase-containingtunable mechanical properties polymers and have self-healing a built-in advantage behaviors, in providing the preparation an efficient of materials potential that route exhibit to tunablethe fabrication mechanical of propertiesmultifunctional and self-healing materials. behaviors, Cheng providing[116] et anal. efficient developed potential a multi-uracil route to the fabricationfunctionalized of multifunctional polyhedral oligomeric materials. silsesquio Chengxane [116] (POSS-U) et al. developed and adenine a multi-uracil end-capped functionalized three-arm polyhedralpolycaprolactone oligomeric oligomer silsesquioxane (PCL-A), (POSS-U) as showed and in adenine Figure end-capped 11. Their three-armmaterials polycaprolactoneshowed a good oligomercomplementary (PCL-A), ability as showed either inin Figuresolution 11 .or Their solid materials state due showed to the uracil a good (U)–adenine complementary (A) cross-linking. ability either inIn solutionaddition, or the solid prepared state duefilm to materials the uracil could (U)–adenine self-heal autonomously (A) cross-linking. at room In addition, temperature the prepared without filmexternal materials intervention, could self-healwhich demonstrated autonomously a simple at room and temperatureefficient way withoutto build a external series of intervention, self-healing whichmaterials demonstrated for a wide range a simple of potential and efficient applications. way to build a series of self-healing materials for a wide range of potential applications.

Uracil-adenine base pairs

Figure 11.11. StructuresStructures of of nucleobase-containing polymers polymers and and an illustration of complementarycomplementary interactions betweenbetween POSS-UPOSS-U andand PCL-APCL-A [116[116].]. ReproducedReproduced withwith permission.permission. Copyright Royal Society of Chemistry, 2015.2015.

Ye et al. al. [117] [117 ]utilized utilized nucleobase nucleobase interactions interactions as asanother another example example of preparing of preparing self-healing self-healing pH- pH-sensitivesensitive biodegradable biodegradable cytosine cytosine and guanosine-mo and guanosine-modiﬁeddified hyaluronic acid hyaluronic hydrogels acid (HA-HMDA-C, hydrogels (HA-HMDA-C,HA-HMDA-G, and HA-HMDA-G, HA-HMDA-C/G) and HA-HMDA-C/G) under physiological under conditions physiological (Figure conditions 12). Their (Figure results 12). Theirindicated results that all indicated materials that have all good materials self-healing have ability, good but self-healing materials ability,with a high but concentration materials with of aHA high had concentrationa short gelation of time HA hadand strong a short mechan gelationical timeproperties. and strong Furthermore, mechanical the mechanical properties. Furthermore,properties of HA-HMDA-C/G the mechanical hydrogels properties were of HA-HMDA-C/Gbetter than those of hydrogels C–C or G–G, were which better confirmed than those that ofthe C–C nucleobase or G–G, interactions which conﬁrmed between that C the and nucleobase G are stronger interactions than the between other two C and types G arein this stronger case. than the other two types in this case.

Polymers 2017, 9, 666 16 of 24 Polymers 2017, 9, 666 17 of 25

FigureFigure 12. 12. IllustrationIllustration andand diagramdiagram of of self-healing self-healing behavior behavior of of (a )(a HA-HMDA-C10,) HA-HMDA-C10, (b) ( HA-HMDA-G10,b) HA-HMDA- G10,(c) HA-HMDA-C/G10,(c) HA-HMDA-C/G10, (d )(d HA-HMDA-C10@HA-HMDA-G10) HA-HMDA-C10@HA-HMDA-G10 hydrogels, hydrogels, andand (e) simulation simulation diagramdiagram of of HA-HMDA-C10@HA-HMDA- [email protected]. The sketches The sketches are of hydr are ofogen hydrogen bonds between bonds cytosine between andcytosine guanosine, and guanosine, with themselves with themselvesor each other, or carried each other, out by carried computer out simulations: by computer (a’ simulations:) cytosines, ((b’a’)) cytosines,guanosines, (b’ and) guanosines, (c’) cytosine and and (c’) cytosineguanosine and [117]. guanosine Reproduced [117]. Reproducedwith permission. with permission.Copyright Elsevier,Copyright 2017. Elsevier, 2017.

4.4.4.4. Self-Assembly Self-Assembly of of Nanoparticles Nanoparticles NanoparticlesNanoparticles (NPs) (NPs) are are among among the the most most distinguished distinguished and and promising promising nanomaterials nanomaterials and and have have beenbeen used used in in a a wide wide variety variety of of applications applications (e.g., (e.g., in in drug drug delivery, delivery, gene gene delivery, delivery, antibacterial, antibacterial, catalysis,catalysis, superhydrophobicity,superhydrophobicity, etc.) etc.) [118 –122[118–122].]. Among Among the many the methods many to fabricatemethods multifunctionalto fabricate multifunctionalNPs for specific NPs purpose, for specific self-assembly purpose, ofself-assembl NPs is identifiedy of NPs as is aidentified crucial and as a efficient crucial and strategy efficient that strategyorganizes that discrete organizes components discrete components into ordered into structures ordered structures by specific by specific interactions. interactions. He [123 He] [123] et al. etprepared al. prepared novel near-infrarednovel near-infrared (NIR) multifunctional(NIR) multifunctional Au–UCNPs Au–UCNPs via nucleobase via nucleobase interaction interaction between betweenCNA polymer-functionalized CNA polymer-functionalized UCNPs UCNPs and complementary and complementary DNA strands DNA strands functionalized functionalized AuNPs AuNPs(Figure 13(Figure). They 13). demonstrated They demonstrated that the conjugation that the conjugation of AuNPs toof UCNPsAuNPs enhancedto UCNPs UCNP enhanced fluorescence, UCNP fluorescence, while AuNSs (nanostars) tended to quench fluorescence. Furthermore, the assembly of

Polymers 2017, 9, 666 17 of 24

Polymers 2017, 9, 666 18 of 25 while AuNSs (nanostars) tended to quench ﬂuorescence. Furthermore, the assembly of AuNSs-UCNP AuNSs-UCNPcould induce rapid could increases induce inrapid temperature increases upon in temperature NIR excitation, upon which NIR providesexcitation, great which potential provides for greatphotothermal potential therapy. for photothermal therapy.

’ ’ ’

Figure 13. 13. TEMTEM images images of the of Au–UCNP the Au–UCNP clusters: clusters: (A) AuNP–UCNP, (A) AuNP–UCNP, (B) 700 (AuNS–UCNP,B) 700 AuNS–UCNP, (C) 980 AuNS–UCNP(C) 980 AuNS–UCNP and (D– andF) the (D –correspondingF) the corresponding optical optical images; images; Images Images of electric of electric field amplitude: ﬁeld amplitude: (A’) AuNPs,(A’) AuNPs, (B’) AuNSs (B’) AuNSs with 700 with nm 700plasmon nm plasmonresonance resonance and (C’) AuNSs and (C’ with) AuNSs 980 nm with [123]. 980 Reproduced nm [123]. withReproduced permission. with Copyright permission. Roya Copyrightl Society Royal of Chemistry, Society of 2015. Chemistry, 2015.

4.5. Metal–Nucleobase Metal–Nucleobase Coordination In nature, metal ions play important roles in the structure and function of nucleic acids. A wide variety of investigations into transition metal ions’ binding to nucleic acids have been utilized extensively, such as in disease diagnostics [124–126]. [124–126]. Other than than applications in in biology, biology, the ability of nucleic acids to store and transmit information has been explored in creating nanostructures and molecular electronic devices [127–129]. [127–129]. Watson Watson et al. [[130]130] introduced 8-hydroxyquinoline (Q) into PNA oligomersoligomers andand investigated investigated the the properties properties of duplexes of duplexes formed formed by these by oligomersthese oligomers in the presence in the presenceof Cu2+. Theirof Cu2+ results. Their demonstrated results demonstrated that the Cuthat2+ -containingthe Cu2+-containing PNA duplexes PNA duplexes have higher have thermal higher thermalstability stability than the unmodiﬁedthan the unmodified PNA, and PNA, furthermore, and furthermore, with the help with of metalthe help ions, of PNAmetal duplex ions, PNA pairs duplexexhibited pairs high exhibited tolerance high to mismatches. tolerance to mismatches. Recently, coordination polymers polymers and and metal metal organi organicc frameworks frameworks (MOFs) (MOFs) ha haveve attracted attracted a lot a lotof attentionof attention from from researchers researchers [131,132]. [131,132 Olea]. Oleaet al. et[133] al. [presented133] presented a simple a simple method method to obtain to obtainsingle polymericsingle polymeric chains from chains crystals from crystalsof [Cd(6-MCP) of [Cd(6-MCP)2·2H2O]n2 ·(6-MCP2H2O]n =(6-MCP 6-mercapto-purinate), = 6-mercapto-purinate), and this particularand this particular 1D MOF exhibited 1D MOF significant exhibited similarities signiﬁcant to similarities DNA-based to systems DNA-based (Figure systems 14). García-Terán (Figure 14). etGarc al.í a-Ter[134] ásynthesizedn et al. [134] asynthesized 3D coordination a 3D coordination polymer using polymer the interaction using the interactionbetween adenine between and adenine Cu2+. X-rayand Cu analysis2+. X-ray indicated analysis indicatedin this covalent in this covalent 3D network: 3D network: the centered the centered Cu2+ Cuions2+ ionsare coordinated are coordinated by tridentate μ-N3, N7, N9 adeninate ligands. This 3D coordination polymer has relatively large, nanometer-sized tubes and exhibited overall antiferromagnetic behavior.

Polymers 2017, 9, 666 18 of 24 by tridentate µ-N3, N7, N9 adeninate ligands. This 3D coordination polymer has relatively large, nanometer-sizedPolymers 2017, 9, 666 tubes and exhibited overall antiferromagnetic behavior. 19 of 25

(A) (B)

P G C P A T P P C G P P T A P P

G O P N NH HN N - π-stacking N S N H2N N II H HN H-bond Cd

FigureFigure 14.14. ((AA)) SchematicSchematic representationrepresentation ofof DNADNA andand [Cd(6-MCP)[Cd(6-MCP)22]]nn.(. (B) Scheme of the structurestructure andand

spaceﬁllspacefill model of of [Cd(6-MCP) [Cd(6-MCP)2]2n] [133].n [133 Reproduced]. Reproduced with with permission permission.. Copyright Copyright John JohnWiley Wiley and Sons, and Sons,2005. 2005.

5.5. Conclusions and OutlookOutlook AlthoughAlthough manymany nucleobase-containingnucleobase-containing polymerspolymers havehave beenbeen synthesizedsynthesized andand appliedapplied in polymer science,science, the the number number of of known known nucleobase-containing nucleobase-containing polymers polymers is still is tiny still compared tiny compared with the with whole the wholepolymer polymer chemistry chemistry community. community. To achieve To achieve a broader a broader impact impact and implementation and implementation in polymer in polymer and andmaterials materials science, science, there are there three are directions three directions to be purs toued. be pursued.Firstly, there Firstly, is still therea huge is need still to a create huge neednovel tonucleobase-containing create novel nucleobase-containing molecular structures molecular including structures monomers, including oligomers, monomers, or oligomers,polymers. orSecondly, polymers. the Secondly, development the development of novel polymeric of novel polymericapproaches approaches for nucleobase-containing for nucleobase-containing polymers polymersremains another remains critical another need, critical especially need, especiallysince most since synthesis most synthesisof nucleobase-containing of nucleobase-containing polymers polymerslacks sequence lacks control, sequence which control, results which in these results nucleobase-containing in these nucleobase-containing polymers not usually polymers exhibiting not usuallysequence-dependent exhibiting sequence-dependent behaviors such as behaviorswe observed such in as nature. we observed Finally, in with nature. the Finally,development with the of developmentpolymer chemistry of polymer itself, chemistry novel and itself, more novel materials and more will materialsbe designed will based be designed on the based nucleobase- on the nucleobase-containingcontaining polymers, which polymers, could which provide could the provide sequence-controlled the sequence-controlled features for features these for“smart” these “smart”materials materials in widespread in widespread applications. applications.

AuthorAuthor Contributions:Contributions: WeixianWeixian XiXi chosechose thethe topic;topic; HaitaoHaitao YangYang and and Weixian Weixian Xi Xi wrote wrote the the paper. paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conflicts of Interest: The authors declare no conflict of interest.

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