engineering and applications of dna-grafted polymer materials

Chemical Science

PERSPECTIVE

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aCenter for Research at the Bio/Nano In

Department of Physiology and Functional

Genetics Institute and McKnight Brain Ins

FL, USAbMolecular Science and Biomedicine Labora

Sensing and Chemometrics, College of Biol

Engineering, Collaborative Innovation C

Medicine, Hunan University, Changsha, 41

[email protected]

Cite this: Chem. Sci., 2013, 4, 1928

Received 7th August 2012Accepted 24th November 2012

DOI: 10.1039/c2sc21198j

www.rsc.org/chemicalscience

1928 | Chem. Sci., 2013, 4, 1928–193

Engineering and applications of DNA-grafted polymermaterials

Lu Peng,a Cuichen Sam Wu,a Mingxu You,a Da Han,a Yan Chen,b Ting Fu,b Mao Ye*b

and Weihong Tan*ab

The emergence of hybrid materials combining biomacromolecules and organic polymers has received

broad attention because of their potential applications in chemical, biological and materials sciences.

Among different coupling strategies, the grafting of oligonucleotides to organic polymers as side chains

by covalent bonds provides a novel platform whereby the properties of both oligonucleotides and

polymer backbone are integrated, manipulated and optimized for various applications. In this review,

we give a perspective on this specific type of DNA polymer hybrid materials using selected examples

with emphasis on bioanalysis, biomedicine and stimuli-responsive materials. It is expected that the

success of DNA-grafted polymers will not only have an impact on the fabrication of novel biomolecule

incorporated materials, but also will influence how the properties of synthetic materials are tailored

using different functional groups.

1 Introduction

A primary goal in synthetic chemistry is the ability to synthesizemacromolecules with well-dened monomer composition andsequence while, at the same time, exhibiting novel propertiesand functions. Materials science and polymer chemistry havebeen extensively used in the generation of articial macromol-ecules with different building blocks, among which bio-origi-nated macromolecules, including proteins, polypeptides andnucleic acids, are of enormous importance.1–4 For decades, thecombination of peptides with organic polymers has beenexplored in depth with a multitude of reviews on their synthesisand supramolecular organization.5,6 However, the coupling ofDNAs with organic polymers has been under-studied.7,8

Owing to technological breakthroughs, the revolutionaryprinciple of solid phase peptide synthesis (SPPS) has beenadapted for the construction of other biomacromolecules suchas DNAs. The solid phase synthesis method enables scientists togenerate DNAs that share similar properties with their naturalcounterparts. Moreover, solid phase synthesis offers a highlyautomated and convenient way to incorporate non-nucleic acidfunctional groups into oligonucleotides which then exhibit

terface, Department of Chemistry and

Genomics, Shands Cancer Center, UF

titute, University of Florida, Gainesville,

tory, State Key Laboratory of Chemo/Bio-

ogy, College of Chemistry and Chemical

enter for Chemistry and Molecular

0082, China. E-mail: [email protected];

8

exciting and unprecedented functions. DNA has been coupledwith various inorganic materials, such as silica nanoparticlesand gold nanoparticles, using different functional groups.9,10

Small organic molecules, including uorescent dyes and pho-toresponsive moieties, have also been incorporated into DNAchains.11,12 Recently, advances in combinatorial chemistry haveenabled widespread research into articial oligonucleotides,particularly the introduction of a novel class of syntheticoligonucleotides known as aptamers. Aptamers are single-stranded oligonucleotides that bind to specic targets and aregenerated from an in vitro process known as systematic evolu-tion of ligands by exponential enrichment (SELEX).13,14 With awide range of targets, including small molecules, proteins, andeven whole cells,15–17 aptamers have found numerous applica-tions in biosensing, molecular imaging, and targeted cancertherapy.18–20 Rapid development in the eld of articial DNA haspropelled its applications in biosensing, biomedicine, andfunctional materials.21–23

During the past decade, a new class of hybrid materials, DNAblock copolymers consisting of oligonucleotides and organicpolymers, has emerged as a major area. Depending on theresulting structures of the hybrid macromolecules, they aredivided into two categories: linear DNA block copolymers andDNA-graed linear polymers. A linear DNA block copolymerconsists of an oligonucleotide and a terminal organic polymer,while a DNA-graed linear polymer is composed of a linearorganic polymer with multiple oligonucleotide strands as sidechains. Different types of moieties have also been incorporatedon one single polymer chain, thus providing new ways for theconstruction of multifunctional materials.24–26 Furthermore,DNA block copolymers have been engineered to form evenmore

This journal is ª The Royal Society of Chemistry 2013

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Perspective Chemical Science

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complex materials, such as multiblock structures, micelles andstimuli-responsive hydrogels.27–30 Therefore, this materials classshows promise in applications ranging from bioanalysis tobiomedicine and materials science. In this perspective,emphasis is placed on DNA-graed linear polymers, andselected examples are given to illustrate the most recent devel-opments in the engineering and applications of DNA blockcopolymer hybrid materials. The perspective is split into threesections: synthesis of DNA-graed linear polymers, directapplications of the DNA-graed linear polymers, and functionalhydrogels assembled from DNA-graed linear polymers. LinearDNA block copolymers are excluded as this eld of research hasbeen extensively reviewed elsewhere.7,8,31,32

2 Synthesis of DNA-grafted linear polymers

DNA-graed hybrid linear polymers feature a synthetic polymerbackbone with multistrand DNAs as side chains to form acomb-like structure. Their synthesis can be realized by twogeneral strategies (Fig. 1). In the rst strategy, DNAs are func-tionalized onto the pre-synthesized linear polymer backbonethrough various types of coupling chemistry (Fig. 1A). One ofthe classic methods includes active ester group-mediatedformation of amide bonds between polymers and DNAs. Forexample, amino-terminated DNAs can be coupled with thecarboxyl groups of poly(acrylic acid) using 1-ethyl-3-(3-dime-thylaminopropyl)carbodiimide (EDC) as a coupling reagent.33

Alternatively, a polymer backbone with activated ester groupscan directly react with amino-modied DNAs to form amidebonds.33 Another method towards DNA modication on pre-synthesized polymers relies on covalent coupling of DNAs withpolymers on a solid support. Phosphoramidite chemistry hasbeen employed starting with the formation of organic polymersbearing multiple phosphoramidite groups. These phosphor-amidite-polymers were then reacted with the detritylated 50-OHend of a DNA on a solid support followed by the cleavage of DNAfrom the support.25,34 Moreover, reactive synthetic polymerswere also immobilized on solid supports for the purpose ofconstructing DNA side-chain polymers.35 In one report, acontrolled pore glass (CPG) surface covered by hydroxyl groupswas covalently coated with an alternating copolymer whichcarried maleic anhydride moieties along the backbone via esterbond formation. Aerwards, the remaining anhydride moieties

Fig. 1 Synthesis of DNA-grafted polymers. (A) Covalent modification of nucleicacids to polymers via coupling chemistry. (B) Copolymerization of polymerizablegroup terminated nucleic acids with polymer monomers.


were reacted to form amide bonds with 50-dimethoxytritylthymidine 30-(6-aminohexyl phosphate) which initiated theoligonucleotide synthesis. The selective cleavage of DNA-graedside-chain polymers aer DNA synthesis was carried out bybasic treatment due to the difference in stability between theester and amide linkages. However, the strategy based on DNAcoupling to presynthesized polymers generally results inconjugates with a small number of DNA strands.

A fundamentally different strategy to synthesize DNA side-chain polymers is based on the copolymerization of polymer-izable DNA monomers with regular polymer monomers such asacrylamide (Fig. 1B). This type of DNA monomer can besynthesized by adding polymerizable moieties onto functionalgroups connected with DNAs. For instance, Murakami andMaeda reported the fabrication of a methacryloyl group-conju-gated ssDNA using methacryloyloxy succinimide and amino-terminated ssDNA.36 Alternatively, polymerizable DNA mono-mers can also be generated directly by solid phase synthesisusing a phosphoramidite having a polymerizable group such asacrydite. Our group has been using acrydite-modied DNAs forthe synthesis of DNA-graed polymer materials for differentapplications.26,37 However, limitations of DNA polymers gener-ated by this strategy include poor solubility and high viscosity,which make them difficult to handle. Another drawback is thatthe resulting structure cannot be fully characterized.

In addition to conventional coupling chemistry for thegeneration of DNA-graed polymers, it is also worthy of atten-tion that click chemistry based strategies have shown greatpromise in the construction of complex macromoleculararchitectures using nucleic acids and other biomolecules. By farthe copper-catalyzed azide–alkyne cycloaddition (CuAAC) reac-tion is one of the most widely used click chemistry approachesbecause of its high efficiency and specicity. CuAAC has beenextensively employed to facilitate the engineering of functionalmacromolecular structures, such as brush and gra copoly-mers, shell or core crosslinked micelles, and hyperbranchedmaterials.38 DNA has also been modied onto organic polymersby this method to create novel macromolecules or assembliessuch as star polymers and micelles.39,40 It is believed that clickchemistry will extend the methodologies for the preparation ofsophisticated DNA–polymer conjugates including DNA side-chain polymers. However, click chemistry-based couplingschemes also have limitations, because excess copper may leadto drastic consequences if products are used for drug delivery.

3 Applications of DNA-grafted hybridlinear polymers

DNA side-chain copolymers are generated by graing DNAsonto the organic polymers allowing the attractive features ofboth materials to be maintained and integrated. For example,both increased melting points and sharp transition are ach-ieved when DNA block copolymers hybridize with complemen-tary DNA block copolymers because of neighboring-duplexcooperativity.41 Compared with mono-strand DNA materials,the assembly of multi-strand DNAs along a single polymer chainintroduces signicant advantages in various applications, such

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Chemical Science Perspective

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as signal-amplied bioanalysis and enhanced therapeuticeffects.

3.1 DNA-graed hybrid linear polymers as sensors for DNAdetection

DNA detection is important in the practice of clinical diagnos-tics, gene therapy, forensic science, and biomedical research.Considerable effort has been put into developing novel mate-rials and methodologies for highly sensitive and selective DNAanalysis. To achieve highly enhanced uorescence signals,various nanomaterials, such as quantum dots, silica and goldnanoparticles, have been investigated as sensing platforms forbioanalysis.42–44 Similar to DNA–nanoparticle conjugates,multiple DNA strands are attached to one single polymer chain.It has been observed that hybridization between two comple-mentary DNA-graed hybrid linear polymers leads to a sharpermelting prole and higher melting temperature than isobserved between two individual complementary strands.41,45 Itis well known that a sharp melting curve is an essential featureof DNA-modied gold nanoparticles and accounts for theenhanced discriminatory power of even single-basemismatches.46 Therefore, because of the arrangement of severalDNAs along the polymer backbone, the cooperative behavior ofDNA hybridization and coupling exibility with multiplesignaling groups has led to signicant improvements in theselectivity and sensitivity of DNA block copolymer-based assays.

DNA detection depends on the vital factors of molecularrecognition and signal readout. In one attempt, Mori andMaeda reported a strategy using DNA-graed poly(N-iso-propylacrylamide) (PNIPAM) colloidal particles.47 In the pres-ence of complementary DNA, destabilization by DNAhybridization on the particle’s surface causes these particles toaggregate, observed as a turbidity increase of the dispersion.However, this method fell short of sensitivity with a detectionlimit in the micromolar range. A second example, as demon-strated by Mirkin and coworkers, took advantage of thenumerous functional groups on DNA block copolymers byadding a number of ferrocene groups as signaling moieties(Fig. 2A).24 The newly constructed triblock conjugates were

Fig. 2 Examples of biosensors for DNA detection based on DNA-grafted poly-mers. (A) Electrochemical detection of DNA employing a DNA–ferrocene–polymerhybrid (adapted from ref. 24). (B) DNA detection using a light-emitting polymerimmobilized on a chip surface (adapted from ref. 48).

1930 | Chem. Sci., 2013, 4, 1928–1938

applied in an electrochemical assay, where the target DNAserved as a bridge to bring the conjugates onto the electrode.Consequently, a redox signal was measured from the ferrocenylmoieties. Both a pM-level limit of detection and single-basemismatch discrimination were achieved. Moreover, dual-channel electrochemical detection of two different DNA targetswas enabled using two distinct block copolymers. Conjugatedpolymers were also employed in the generation of DNA blockcopolymers for DNA analysis (Fig. 2B).48 On-chip DNA synthesiswas carried out on a poly(oxadiazole-uorene) derivative poly-mer-coated glass slide to fabricate a signal-amplifying DNAchip. Upon the hybridization of dye-labeled target DNA with theDNA graed on the conjugated polymer, uorescence reso-nance energy transfer (FRET) from the conjugated polymer tothe dye on the target yielded a uorescence readout for detec-tion. Furthermore, the chip-based assay can be performed withease in a parallel fashion, which has the potential for devel-oping a high-throughput method. However, up to now, methodsbased on DNA-graed polymers generally have a relatively highlimit of detection. To further improve the performance, it isnecessary to introduce signal amplication via approaches suchas enzymatic reactions. Moreover, the capability for single-basemismatch discrimination is still lacking. More sophisticatedprobe designs are expected in the future.

3.2 DNA-graed hybrid linear polymers used in DNA delivery

DNA not only provides the molecular basis for the under-standing of the biology of human life but also serves as afundamental building block for the construction of noveltherapeutics. Functional DNA-based therapeutics include plas-mids for gene therapy, antisense oligonucleotides, DNAzymesand aptamers. However, the therapeutic applications of DNAhave been limited due to poor cellular uptake and rapid enzy-matic degradation. Different methods and delivery systemshave been developed to facilitate cellular internalization ofDNA-based therapeutics with maintained activity.49,50

DNA-graed polymers also nd unique applications in thisarea. For a few decades, polymer-based therapies have gainedattention as a result of their potential advantages over conven-tional drugs, including biocompatibility, effectiveness intransmembrane delivery and exible integration with chemo-therapies.51,52 It is well known that positively charged polymerscan be effectively internalized into cells, thus serving as effectivecarriers for various anticancer drugs. Lebleu and coworkersengineered a DNA-modied poly(L-lysine) (PLL) polymer

Fig. 3 (A) Structure of DNA–poly(L-lysine) conjugate (adapted from ref. 53). (B)Temperature controlled binding of DNA–PNIPAM conjugate with target mRNA(adapted from ref. 55).




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(Fig. 3A).53 This type of DNA polymer hybrid can be more easilytransported into cells than pristine DNAs. More interestingly, a15-mer antisense DNA-graed PLL polymer targeting the initi-ation region of the mRNA of vesicular stomatitis virus (VSV)N-protein specically inhibited expression of VSV proteins at adosage as low as 100 nM. Moreover, a similar approach wasemployed to deliver antisense oligonucleotides into humanimmunodeciency virus type 1 (HIV-1) for inhibition of virusreverse transcription.54

Poly(N-isopropylacrylamide) (PNIPAM) is a temperature-responsive polymer regarded as one of the most well-knownintelligent polymer materials. Maeda and coworkers reportedan antisense DNA–PNIPAM side chain polymer with tempera-ture controlled antisense activity (Fig. 3B).55 This DNA–PNIPAMconjugate could specically bind a complementary RNAsequence at 27 �C below the lower critical solution temperature(TLCST, 33 �C) when the conjugate stayed in random coil struc-ture. However, as the temperature was raised to 37 �C aboveTLCST but still below the DNA melting temperature, coil-to-globule transition of the PNIPAM backbone signicantlyinhibited the binding of antisense DNA with the target. Theconjugate was later applied in an in vitro study: gene expressionat 27 �C was inhibited at a similar level to that when pristineDNA was used, while at 37 �C, no signicant inhibition effectwas observed.56 Overall, DNA delivery using DNA-graedpolymers possesses biological potential, but a systematicunderstanding is still needed to interpret the behavior of theDNA-graed polymers in the transmembrane delivery, intra-cellular fate, biocompatibility, etc.

Fig. 4 Aptamer-grafted acrylamide polymers for selective cytotoxicity. (A)Scheme of polymeric aptamers. Selective cytotoxicity was achieved with (B) non-drug-resistant cancer cells and (C) drug-resistant cells (adapted from ref. 26).

3.3 DNA-graed hybrid linear polymers used in cancertherapy

Anticancer therapeutic drugs to inhibit cellular proliferation arelimited by their general toxicity. Although anticancer drugswork more intensely on a particular type of cancer cell, ratherthan on healthy cells, some side effects always occur throughoutthe systematic dosing process. Consequently, different types ofnovel therapeutic agents have been developed with distinctmechanisms. However, specic selectivity of these therapeuticstoward cancer cells is still lacking, and none has been clinicallyuseful. Monoclonal antibodies offer an alternative strategy forselective cancer therapy with lower toxicity; but, they fall shortdue to availability problems and their modest effects. Similarly,the development of multidrug resistance, another challenge inthe eld of cancer therapeutics, is driving advances in target-specic drugs with enhanced efficacy.57,58

In the pursuit of better anticancer therapeutics, muchattention has been focused on the engineering of new thera-peutic hybrid materials using anticell aptamers selected usingthe method known as cell-based systematic evolution of ligandsby exponential enrichment (Cell-SELEX).17,59 Aptamers withspecial properties have been identied, such as Sgc8, which canbe internalized by specic cancer cells. Owing to the exibilityof solid phase synthesis, different types of functional groupscan be modied on aptamers. Combining aptamers and poly-mers, a targeted anticancer system was developed by using the


targeting property of aptamers and the intrinsic toxicity ofpolymers (Fig. 4A).26 The hybrid polymer was synthesized bycopolymerization of acrylamide, acrydite-modied Sgc8aptamer, and acrydite/FITC-modied reporter DNA monomer.The resulting polymers exhibited high specicity and internal-ization ability based on the coupling of the Sgc8 aptamer.Furthermore, the DNA polymer hybrid induced selective cyto-toxicity in target cell lines, while having little effect on non-target cells (Fig. 4B). Furthermore, when the aptamer-polymerconjugate was tested with the drug-resistant K562/D cell line,signicant cytotoxicity similar to that of the correspondingnondrug-resistant K562 cell line was observed (Fig. 4C). Theresults indicated that the aptamer–polymer conjugate couldbypass the P-glycoprotein (P-gp), a drug efflux transporter, onthe cell membrane of the drug-resistant K562/D cells andinterrupt cellular metabolism. Therefore, this approach maynd potential applications in the development and improve-ment of anticancer drugs, as well as in the targeted delivery of

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cancer therapies. However, the exact mechanism of the thera-peutic effect remains unclear although further studies showedthat the signicant cytotoxicity of the DNA-graed poly-acrylamide conjugates is likely rooted in the intrinsic propertiesof the polyacrylamide backbone, including physical size, pres-ence of primary amine groups, and exibility.

4 DNA-crosslinked dynamic polymerhydrogels

A hydrogel is a network of crosslinked hydrophilic polymers.Hydrogels can hold large amounts of water, up to more than99% of their weight. Stimuli-responsive dynamic hydrogels haveshown great potential in various applications, such as bio-sensing, drug delivery and microuidics. Based on the differentdesigns, several classes of hydrogels have been developed withsensitivities towards temperature, pH, biomolecules, and otherstimuli.60–63 These dynamic hydrogels can change such proper-ties as volume, color and transparency in response to environ-mental stimuli.64,65

Towards the development of novel functional hydrogelmaterials, various types of molecular interactions have beenintroduced into the polymeric hydrogel systems, includingantibody/antigen binding and conformational change ofproteins.66,67 The DNA-graed polymers can also be furtherassembled to form hydrogels, mainly through two differentstrategies. The rst involves addition of permanent linkergroups to induce the covalent-bond crosslinking of polymerbackbones where the DNAs are also modied. In this strategy,either a dual-functionalized ssDNA, such as ssDNA with aminogroups on both ends, or other types of molecules, such as N,N0-methylenebisacrylamide (MBAm), serve as crosslinkers.36,68 Inthe second approach, duplex DNAs formed by DNA hybridiza-tion serve as the crosslinking groups for the hydrogels. Thereversible nature of DNA hybridization in response to externalstimuli causes these types of DNA polymer hydrogel to havespecial properties, such as sol–gel phase transitions andresponsive releasing capabilities. As a result, a number ofsystems with different applications have been developed basedon this class of hydrogels.

Fig. 5 (A) Target ssDNA induced swelling of a semi-IPN DNA-grafted polymerhydrogel (adapted from ref. 68). (B) Target ssDNA induced volume change ofhairpin DNA (top) and DNA without secondary structure crosslinked polymerhydrogels (adapted from ref. 36).

4.1 DNA-graed polymer hydrogels for sensor applications

Stimuli-sensitive hydrogels that can sense environmentalchanges and undergo structural changes hold great promise inthe development of chemical sensors.69,70 For example, somesensitive hydrogels can swell in response to specic biomole-cules, such as proteins and glucose, and have become impor-tant in the development and application of responsivebiomaterials and smart drug delivery systems.66,71 A ratiometricuorescent pH sensor has also been developed based on reso-nance energy transfer caused by the volume change of chitosanhydrogels.72

DNA sensing hydrogels have been fabricated using volumechange as the output. Maeda and Murakami synthesized apolyacrylamide hydrogel containing DNA-graed polymerconjugates in a semi-interpenetrating network (semi-IPN)

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manner (Fig. 5A).68 The addition of target DNA caused DNAhybridization resulting in shrinking of the hydrogel. Althoughthe exact mechanism was still unclear, one possible explanationwas proposed that dehydration occurs when the duplex DNAforms, and water molecules diffuse out of the hydrogel. UsingDNA as crosslinkers, the same group designed a new DNA-responsive hydrogel structure (Fig. 5B).36 A DNA–polymer hybridhydrogel was prepared by copolymerization of acrylamide andssDNA modied with methacryloyl groups on both the 30 and 50

ends. When the DNA crosslinker was designed as a hairpin, thehydrogel swelled upon the addition of complementary DNA.This volume increase was explained by the hybridizationinduced elimination of the stem–loop structure and longitu-dinal extension of the DNA crosslinkers. In contrast, when theDNA crosslinker was designed with no secondary structure, avolume shrinkage was obtained due to the addition of thecomplementary ssDNA. For the DNA crosslinked hydrogels, thevolume change behavior largely depends on the structure ofthe DNA crosslinkers which can lead to the rational design ofhydrogels with desired responsiveness for DNA sensing appli-cations. However, the application is limited by the slowresponse rate and small percentage volume change. Furtherefforts are necessary to optimize the structural properties of thistype of DNA-crosslinked hydrogel.

Beside the DNA responsive hydrogels, small moleculeresponsive hydrogels have also been engineered based onaptamer–target interactions (Fig. 6A).73 These dynamic hydro-gels undergo a gel-to-sol transition in the presence of a specictarget. The basic principle of operation relies on a



Fig. 6 (A) Detection of adenosine using aptamer crosslinked hydrogels by sol–gel transition (adapted from ref. 73). (B) Signal amplified detection of targetsbased on colorimetric reaction catalyzed by enzymes released from target-responsive hydrogels (adapted from ref. 77). (C) Detection of mercury ions usingthymine-rich DNA-grafted polymer hydrogels (adapted from ref. 78).


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target-induced decrease in duplex DNA-crosslinker density ofthe hydrogel. In order to construct this hydrogel, two acrydite-modied DNA strands, strand A and strand B, are separatelycopolymerized with acrylamide to generate DNA-graed poly-mers A and B in solution. A linker ssDNA containing an aptamersegment is then added to form the hydrogel. The sequenceswere designed such that the three-strand DNA complex woulddissociate in the presence of target due to a target-inducedstructural change of the aptamer. Consequently, the hydrogelconverts back to the solution state if a signicant amount oftarget is added. In this detection approach, the target-respon-sive sol–gel phase transition could be visually detected, and thedetection of adenosine and thrombin as model targets wasdemonstrated.

Although direct visual detection based on the gel-to-solconversion of DNA polymer hydrogels can be used for bio-sensing, the detection limit is poor, and quantitative analysis isdifficult to achieve. Moreover, most biosensing systems basedon the structural changes of hydrogels are unsuitable for prac-tical applications.74–76 Recently, colorimetric assay-based visualdetection methods have provided a simple and rapid, yet cost-effective, method for sensitive biosensing as a result of theemergence of aptamers. In order to improve detection usingtarget-responsive hydrogels, a novel visual detection systemusing colorimetric agent-caging hydrogels has been developed(Fig. 6B).28 Similar to the system described above, two DNA-graed polymers were synthesized separately. Before the addi-tion of aptamers to induce gelation, agents for signal generationwere mixed with the DNA polymers. For colorimetric detection,gold nanoparticles were added, and the upper solution turnedred when target was present. As a further step, better analytical


performance was achieved when an enzyme, amylase, wasintroduced instead. It is well known that amylose induces acolor change of iodine solution from yellow to dark blue andthat amylase converts amylose into sugar. In this hydrogelsystem, the presence of targets caused the release of amylaseinto the dark blue solution containing amylose and iodinewhich changed color to yellow as a readout. Overall, thiscolorimetric agent–caging hydrogel system showed goodsensitivity and high specicity.

Additionally, Liu and coworkers described a strategy using athymine-rich DNA-modied polyacrylamide hydrogel to detectand remove mercury ions from water simultaneously (Fig. 6C).77

Detection relied on the formation of hairpin structures inducedby Hg2+ mediated T–T DNA base-pairing in the hydrogel. TheT–Hg2+–T base pair has a higher stability than the classical T–Apair and cannot exist with ions other than Hg2+.78 Aer theformation of hairpin in the presence of Hg2+, SYBR Green I, acyanine dye that emits green uorescence on binding to duplexDNA, is added to the hydrogel. Green uorescence wasobserved. However, in the absence of Hg2+, yellow uorescencewas obtained. A detection limit of 10 nM Hg2+ by visual detec-tion was achieved with a 50 mL water sample. Moreover, theadsorbed Hg2+ could be removed by acid treatment for theregeneration of the hydrogel.

The above examples demonstrate the applications of DNA-graed hydrogels for detection. But currently, the performanceof this class of hydrogels is limited by some intrinsic properties.First, the response rate is slow compared with solution baseddetection methods due to the restricted diffusion of moleculesin the hydrogel network. Second, quantitation by this strategy ishard to achieve. For example, the volume change or sol–gelconversion cannot be directly related to the target concentra-tion. Although the introduction of release of signaling groupsprovides well-measurable signals, accurate quantitation stillremains a problem due to experimental variations.

4.2 DNA-crosslinked polymer hydrogels designed for logicgates

Aside from the utilization of DNA crosslinked polymer hydro-gels for biosensor development, other new functions are alsobeing explored. The engineering of molecular logic gate systemsthat generate output signals in response to chemical andphysical inputs has attracted extensive attention. Recently, anew class of DNA-based molecular logic gates has shown specialfeatures and is considered an excellent platform for in vitrocomputation.79,80 DNA-graed polymer hydrogels have emergedas one of the promising platforms for logic gate systems.

An example of this type of logic gate system is involved in thesynthesis of polyacrylamide hydrogels with both permanentand duplex DNA crosslinkers. Depending on DNA hybridiza-tion, the hydrogels transformed DNA based logic operationsinto a volume change of the hydrogel.81 As illustrated in Fig. 7,two DNA strands with two isolated complementary segmentswere modied on the hydrogel polymers to form the duplexDNA crosslinkers. The swelling behavior of the hydrogels couldbe adjusted by adding different complementary DNAs which

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Fig. 7 Illustration of hydrogel swelling behavior in response to complementarysequences monitored by the interferometric technique (adapted from ref. 82).

Fig. 8 Colorimetric logic gates based on aptamer-crosslinked hydrogels: (A)AND gate and (B) OR gate using cocaine and ATP as stimuli (adapted from ref. 83).


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induced partial or complete dissociation of the duplex DNAcrosslinkers through toehold displacement reactions. Thedegree of volume change was evaluated by interferometrictechniques using height as the output. Logical AND and ORoperations were realized based on the volume swelling levels.When a single complementary DNA (P1 or P2) was added,breaking one of the two duplex segments of the DNA cross-linker, the hydrogel swelled to a level represented by the heightL1. At the level L1, the OR gate was realized with the signal “1”when either P1 or P2 was present. Furthermore, a morepronounced signal (L2) was achieved when both P1 and P2 wereadded to the hydrogel to completely disintegrate the duplexDNA crosslinkers. Therefore, at the level L2, the AND gate wasrealized with the signal “1” in the presence of both P1 and P2.The reason for the different swelling behaviors upon addition ofdifferent complementary DNAs was attributed to the corre-sponding Donnan effect on the polyelectrolyte hydrogelswelling balance and change in crosslinker density.

Inspired by the target-responsive DNA polymer hydrogels,extra target recognition functions were introduced to the systemin order to construct colorimetric logic gates that respond tochemical stimuli.82 Three ssDNA strands containing aptamersegments were involved in the self-assembly of responsive DNAnanostructures. Two logic gates (AND and OR) for cocaine andATP were fabricated. In the AND gate (Fig. 8A), a hydrogelloaded with output gold nanoparticles (AuNPs) was produced bycrosslinking two DNA-graed polymers through a three-wayDNA junction when a linker DNA was added. The DNAsequences were designed such that the presence of a singletarget only could disintegrate one arm of the three-way junction.Therefore, the DNA polymers were still crosslinked to maintainthe hydrogel structure resulting in no release of AuNPs (output“0”). However, the hydrogel AND gate yielded an output “1” onlywhen both targets were added to destroy the three-way junctionstructure.

In the design of the OR gate (Fig. 8B), the linker DNA bridgedthe two DNA-graed polymers to form the hydrogel. Moreover,the sequence of the linker DNA was designed to contain twotoehold aptamer segments on both termini for binding withtheir respective targets. The presence of either cocaine or ATPcould cause the breakage of the crosslinkers by the aptamer–target induced dissociation of duplex DNA. Therefore, the logicAND gate was realized to yield an output “1” upon the additionof either target. Since the logic gate hydrogel system canperform Boolean operations in response to different molecular

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inputs, molecular computation is a practical outcome. More-over, this type of multi-input hydrogel system has potentialapplications in diagnostics and biomedicine, especially whenmedical conditions are determined based on combinations ofbiomarkers. On the other hand, the described hydrogels weresynthesized at the macroscopic level which is not sensitive tothe logic input under a micro-environment. In addition, theslow response of the hydrogels may also limit their applications.Future studies are need to scale down the logic DNA hydrogelsand improve the diffusion properties within the network.

4.3 DNA-crosslinked polymer hydrogels for controlled drugdelivery

Drug delivery systems with temporal and spatial control arehighly desired in the development of next-generation thera-peutics with excellent efficacy and specicity, and minimal sideeffects. Responsive polymer materials have been intensivelyinvestigated for controlled drug delivery, and smart polymerhydrogels have received enormous attention.83,84 DNA-graedpolymer hydrogels provide a versatile platform with the poten-tial for different biomedical applications.7,8 Integrated withdifferent functional groups, the structures and functions ofnucleic acids can be easily tuned with various stimuli, such aschemicals, pH, or light, to form stimuli-responsive DNAhydrogels for controlled drug delivery and release.85–87




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In one report, Simmel and co-workers demonstrated a DNA-controlled hydrogel release system using quantum dots (QDs)as a model drug (Fig. 9A).88 The DNA hydrogel was synthesizedusing a similar method to that described above.73 The DNAcrosslinker strand had a toehold that could recognize therelease DNA. When complementary release DNA strands wereadded to the system, toehold initiated strand displacementcaused the hydrogel to dissolve followed by the release of QDsfrom the network. Aerwards, the hydrogel could be regen-erated by adding crosslinker DNA strands again. This type ofduplex DNA crosslinked hydrogel is biocompatible andprogrammable. The properties, such as mechanical strength,pore size and melting temperature, can be adjusted by thelength, sequence design and concentration of DNA strandsemployed during the hydrogel synthesis. Furthermore, theintegration of aptamers will facilitate the development ofcontrolled delivery systems responsive to a broad range oftargets.

In addition, stimuli other than chemicals were also appliedto trigger cargo release from DNA hydrogels. A photoresponsiveDNA-crosslinked hydrogel that undergoes reversible sol–gelconversion upon irradiation with UV and visible light was

Fig. 9 Controlled release from DNA-crosslinked hydrogels. (A) DNA inducedrelease of QDs (adapted from ref. 89). (B) Light controlled release from a pho-toresponsive DNA-crosslinked hydrogel (adapted from ref. 29). (C) NIR lighttriggered thermal release from DNA nanogels coated on gold nanorods (adaptedfrom ref. 37).


created as shown in Fig. 9B.29 In the synthesis of the hydrogel,an azobenzene modied DNA crosslinker which hybridizedsimultaneously with both DNA strands graed on the polymerswas added instead of a regular DNA crosslinker. The azo-benzene-regulated DNA hybridization was responsible for thereversible sol–gel conversion. Specically, the UV/visible lightirradiation drove the isomerization of azobenzene moietiesbetween the trans and cis states, thereby regulating the associ-ation and dissociation of complementary ssDNA.89,90 Theencapsulation and release of small molecules, proteins, andnanoparticles was tested in a light-controlled fashion. Theresults showed that signicant release occurred only uponirradiation with UV light. The biocompatibility of the hydrogelswas also veried in cell experiments, which exhibited limitedtoxicity. Furthermore, the biomedical applications weredemonstrated using the photoresponsive DNA hydrogel forcontrolled release of an anticancer drug, doxorubicin, to killcancer cells.

As shown in the previous example of controlled release, DNA-crosslinked hydrogels show impressive capability. However,theymay not be optimal when the targeting region is deep in thehuman body or when the stimuli are subtle, i.e., weakbiochemical signals or subnanomolar-level biomarkers. Onefeasible approach is to design hydrogels that respond toexternal stimuli, such as light or magnetic and electrical elds.Another approach is to engineer nanometre-sized deliverysystems that are sensitive to localized weak stimuli. Goldnanoparticles can absorb light energy in the near-infrared (NIR)range and generate heat to cause an increase of temperature inthe surrounding area which can be used for manipulation ofheat-sensitive events, such as DNA hybridization and celldestruction.91,92 Based on photothermal nanoparticles and aDNA-crosslinked hydrogel, a NIR light-responsive core–shellnanogel for targeted drug delivery was developed (Fig. 9C).37

This nanostructured hydrogel particle was constructed from ananosized DNA-crosslinked polymer hydrogel and anembedded gold/silver nanoparticle with drug molecules trap-ped in the hydrogel layer. Cell-specic aptamers were alsomodied on the hydrogel network to guide the therapeuticnanogels to the targets. Upon irradiation with NIR light,elevated temperature caused the DNA-crosslinked hydrogellayer to undergo a rapid melting process by the dissociation ofDNA duplex. Meanwhile, the payload drugs were released togenerate a therapeutic effect. The NIR light-controlled release oftherapeutics was investigated using doxorubicin on cancercells. The results showed that the drug-loaded targeted NIRlight-responsive nanogel possessed high therapeutic effect withspecicity toward target cancer cells.

As for the hydrogels mentioned in the previous sectionssharing the same gel-to-sol conversion, this class of stimuli-responsive hydrogel delivery system may suffer from their largesize which results in problems including insensitivity to stimuli,lack of accurate control over release and nonspecic leakage ofdrugs. The azobenzene functionalized DNA hydrogel offers away of using light as the external stimulus of which the dosageand position can be better administered. But the required UVlight has limited tissue penetration capability and is harmful to

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the human body. The NIR light responsive nanogel partiallysolves the problem with extra targeting function. Futureresearch may include the optimization of hydrogel propertiesand the development of DNA hydrogel release systems respon-sive to other stimuli such as pH, electric and magnetic eld.

4.4 DNA-crosslinked polymer hydrogels exhibitingphotoreversible volume changes

Stimuli-responsive hydrogels as platforms for the encapsulationof sensing materials and therapeutic drugs have signicantapplications in bioanalysis, diagnostics and biomedicine, butthe physical changes of hydrogels responding to externalstimuli can also be utilized in the engineering of novel dynamicdevices, such as actuators and microlenses. This type ofhydrogel can performmechanical operations, including volumechange and deformation.93,94 Depending on their design,dynamic hydrogel devices are able convert different types ofenergy, including chemical energy, electrical energy and heat,into mechanical movement.

Our group has engineered a novel hydrogel with reversiblevolume changes based on light-regulated DNA hybridization(Fig. 10).30 Photoresponsive hydrogels have attracted enormousattention.64,95 Light is one of the most attractive stimuli forhydrogels since it can be delivered remotely with controlledintensity and high accuracy. Moreover, light-responsive mate-rials are increasingly studied because of their potential in solarenergy harvesting and utilization.96,97 In our design, azo-benzene-regulated photoswitchable DNA duplex complexeswere graed into the hydrogel network as reversible cross-linkers. A two-step polymerization method was used, resultingin a hydrogel in which an azobenzene-modied DNA-graedlinear polymer was entrapped in a complementary DNA-graedcrosslinked polymer network. The hydrogel was maintained bypermanent N,N0-methylenebisacrylamide (MBAA) crosslinkers,while the azobenzene-modied DNA duplex complexes servedas reversible crosslinkers. Therefore, the overall hydrogelcrosslinker density could be reversibly manipulated by UV andvisible light. Upon UV light irradiation, the DNA crosslinkerswere dissociated, yielding a larger volume of the hydrogel, whileirradiation with visible light caused the recovery of the DNA

Fig. 10 (A) Light-controlled DNA hybridization. (B) Reversible volume transitionfrom ref. 30).

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crosslinkers, resulting in a smaller volume. The process ofvolume transition was reversible when UV and visible light wereapplied alternately. The volume change of the hydrogel couldpotentially be (1) converted into mechanical or electrical energyif proper device designs are used or (2) extended to similarphotoresponsive systems, such as azobenzene/a-cyclodextrincomplexation. On the other hand, there is still plenty of roomfor improvement of this type of hydrogel. The response rate isrelatively slow due to the large size and the restricted diffusion.Possible strategies to further enhance the responsivenessinclude the utilization of a porous structure and the fabricationof micro-sized hydrogel particles.

5 Summary and outlook

Synthetic polymers have been the focus of materials science andchemistry since they rst appeared because of their majorimpact on scientic research and daily life. Nucleic acids,known as the molecular foundation of all life forms, have foundnew applications in areas other than genetics, such as materialsscience, nanoscience and combinatorial science. Thanks todevelopments in the eld of articial nucleic acids, verydifferent functions can be integrated into one system, whichexpands our toolbox of functional nucleic acids. Specically,hybrid materials combining nucleic acids and synthetic poly-mers provide us with special features that are not observed ineither material alone. Various methods have been developed tosynthesize DNA polymer hybrid materials with novel propertiesand applications. As discussed in this perspective, DNA-graedlinear polymers have been successfully applied in bioanalysisand biomedicine. Furthermore, this class of polymers supportsthe formation of more complicated structures, such as cross-linked hydrogels to assist DNA hybridization. Applications inbiosensor development, logic gate fabrication, controlled drugrelease and novel photodynamic materials have been explored.However, their physical and chemical properties need moreintensive investigation in order to extend their applications.Moreover, to understand the mechanisms underlying theirtherapeutic effects, the interaction of DNA-graed polymerswith cells also requires systematic study. Similarly, the stimuli-responsive release process of DNA hydrogels should be

of the DNA-crosslinked hydrogel regulated by UV and visible light (adapted




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elucidated. As our knowledge of DNA-graed polymers deepensand broadens, the properties will be better controlled for morepractical applications.

Acknowledgements

The authors would like to thank Dr Kathryn R. Williams formanuscript review. This work is supported by grants awarded bythe National Key Scientic Program of China (2011CB911000),the Foundation for Innovative Research Groups of NSFC (Grant21221003), China National Instrumentation Program2011YQ03012412 and by the National Institutes of Health(GM066137, GM079359 and CA133086).

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