Direct DNA Conjugation to Star Polymers for ... - ACS Publications

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Direct DNA Conjugation to Star Polymers for Controlled Reversible Assemblies Saadyah Averick,# Eduardo Paredes,# Wenwen Li, Krzysztof Matyjaszewski,* and Subha R. Das* Department of Chemistry and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States

bS Supporting Information ABSTRACT: Polymer biomolecule hybrids represent a powerful class of highly customizable nanomaterials. Here, we report star-polymer conjugates with DNA using a “ligandless” Cu(I) promoted azidealkyne cycloaddition click reaction. The multivalency of the star-polymer architecture allows for the concomitant conjugation of other molecules along with the DNA, and the conjugation method provides control over the DNA orientation. The star-polymer DNA nanoparticles are shown to assemble into higher-order nanoassemblies through hybridization. Further, we show that the DNA strands can be utilized in controlled disassembly of the nanostructures.

’ INTRODUCTION The marriage of synthetic polymers with naturally occurring macromolecules has led to offspring that display complex biomacromolecular architectures thereby revolutionizing the field of bioconjugations.16 Bioconjugates can be obtained from copolymers produced through atom transfer radical polymerization (ATRP), as well as other controlled radical polymerization (CRP) procedures, such as reversible additionfragmentation chain transfer (RAFT) and nitroxide-mediated polymerization (NMP).712 One of the key features of these CRP procedures is the ability to produce copolymers with well-defined polymer architectures, including linear, brush, and star-shaped molecules, with a high content of functional chain ends.6,1318 The polymer chain ends have recently been used in further conjugations to install biotin for avidin binding,1921 carbohydrate conjugates for lectin binding,22,23 or direct proteinpolymer hybrids.4,2436 A tetrafunctional RAFT initiator used in a “core-first” method of star synthesis yielded a tetrafunctional maleimide star polymer that was conjugated to lysozyme, generating a multiprotein star polymer hybrid. In another example, tert-butyl acrylate was polymerized from a tetra-functional ATRP initiator and its bromochain ends were substituted by reaction with sodium azide to produce azide-terminated star polymers that were conjugated to a peptide.24 The ease of obtaining multivalent functional polymeric architectures by CRP techniques has enriched the field of bioconjugates and has the potential to lead to further novel materials. Polymeroligonucleotide hybrids, a relatively new class of biomacromolecules, are bioconjugates of significant promise. Maynard et al.37 conjugated siRNA to a thiolated chain of a poly(oligo(ethylene oxide)methacrylate) prepared using RAFT. The disulfide linkage formed in this conjugate was labile under r 2011 American Chemical Society

reducing conditions. A more robustly linked polymerDNA hybrid was prepared from polymers synthesized using ATRP.38 DNA synthesized on beads was directly coupled to a phosphoramidite-terminated polystyrene that has, in previous research, been used as an amphiphilic DNA block copolymer to encapsulate nanoparticles.39 Reaction of maleimide chain ends yielded thioester-conjugated DNA aptamers. The aptamers, or singlestranded DNA sequences that bind small-molecules, retained in vitro functionality in binding to their target peptide. Proposed applications for these polymeroligonucleotide (DNA or RNA) hybrids have mainly been therapeutic stabilization and delivery of detection agents. However, DNA can provide other functional and structural properties, in vitro, and in vivo,4042 that can be applied advantageously to polymer hybrids. PolymerDNA conjugates would thus provide nanoscale macromolecules that could be controlled using sequence-specific hybridization and other functional properties of DNA that are highly tunable (Scheme 1). DNA self-assembly and nanotechnology are well-developed fields and DNA can be used to create three-dimensional materials such as nanoscale objects,4345 macroscopic crystals,4648 and DNA-based gels.49 DNA self-assemblies and origami50 are now moving from static to dynamic systems.51,52 However, although those designs are based solely or largely on DNA, ready access to large (nanoscale) three-dimensional objects and their manipulation, remains challenging. Large colloidal metal-based nanoparticles can be improved in their utility with a functional coating of DNA.5360 Although nanoparticleDNA hybrids and nanoscale Received: May 6, 2011 Revised: August 18, 2011 Published: August 26, 2011 2030

dx.doi.org/10.1021/bc200240q | Bioconjugate Chem. 2011, 22, 2030–2037

Bioconjugate Chemistry

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Scheme 1. Strategy for Star PolymerDNA Hybrids by Direct Conjugationa

a

These nanostructures would have high tunable control over design parameters. The star polymerDNA conjugate would combine useful characteristics of each material into one molecule.

objects are both easily prepared and are useful, the DNA coating is nonspecific and the ability to custom tailor the coat and core is limited. A polymer nanoparticle would provide a highly customizable core material when used with DNA, including the possibility of concomitant molecules along with the DNA coat through the use of an appropriate conjugation methodology. One of the most convenient conjugation strategies is the copper-catalyzed azidealkyne cycloaddition (CuAAC) reaction.61,62 This highly efficient reaction has risen to preeminence among the various “click” reactions63 because the reacting azide and alkyne groups do not cross react with biologically abundant functional groups. The CuAAC reaction thus enables robust bio-orthogonal conjugations in aqueous media.64,65 This click reaction has been extensively applied to the synthesis of polymers66,67 and proteinpolymer hybrids.1,4,9,68 Besides the use with polymers and biomolecules, particularly proteins, click chemistry has been useful with synthetic and biochemically obtained DNA. DNA can be obtained with a high density of alkyne groups, and alkyne-modified DNA has been used with CuAAC for a wide variety of applications.54,6972 Although CuAAC has seen widespread use in both polymer chemistry and DNA supramolecular chemistry, the convergence of polymeric material and DNA through click conjugations is limited.67,7375 We show that, by using CuAAC, ATRP based multivalent star polymers can be readily conjugated to functionalized DNA. Because click chemistry is used, simultaneous conjugation of these multivalent polymer cores with DNA, as well as other molecules, yields multicomponent-armed hybrids. We show that a polymer core armed with DNA provides a novel nanoscale material that can be designed quite simply, such that the DNA arms of the star can be used to engender function. While DNA can provide numerous useful functions, as a simple proof of principle for star-DNA hybrids, we demonstrate the use of hybridization properties of DNA to control the size of the star polymer nanoparticles. The self-assembly of star-DNA particles can be controlled and displacement with cDNA can reverse the assemblies. This preliminary study expands the scope of polymerDNA hybrids by demonstrating how loading of DNA onto a multivalent architecture can provide control over selfassembly of polymer nanoparticles.

’ RESULTS AND DISCUSSION Click Conjugations of DNA and Concomitant Molecules onto Star Polymers. Multiarm star polymers with peripheral

Table 1. DNA Sequences Used in This Study terminus name

50 -

sequence

30 -

DNA1

50 -cgc aag aag agc aaa cgc

Dy547

O-propargyl

DNA2 DNA3

50 -gcg ttt gct ctt ctt gcg 50 -ggc cga cgt gct tcg gct cgt

Dy547 phosphohexynyl

O-propargyl OH

DNA4

50 -aat taa cga gcc gaa gca cgt

phosphohexynyl

OH

DNA5

50 -acg agc cga agc acg tcg gcc

OH

OH

functional azide-terminated arms were prepared by the macroinitiator “arm first” method. The macroinitiator was prepared using 3-azidopropyl 2-bromophenylacetate as an ATRP initiator for polymerization of OEO300MA (oligo(ethylene oxide) methacrylate with Mn = 300) targeting a degree of polymerization DP = 49. The macroinitiator was then copolymerized with ethylene glycol diacrylate (EGDA) under dilute conditions. The star polymers were analyzed using multiangle laser light scattering (MALLS) and determined to have Mn = 122 100, corresponding to average 7 arms per star. These star polymers with azideterminated arms were conjugated with several different DNA strands that bore a reactive alkyne using click chemistry. Synthetic sequences of DNA with an alkyne, either at the 50 - or 0 3 -terminus, were obtained using commercially available reagents and protocols recommended by the manufacturer. For these studies, several DNA sequences were prepared with either a 50 phosphohexynyl or 30 -O-propargyl group. In addition, some of the DNA strands included a terminal fluorescent dye that was incorporated during solid-phase synthesis using a commercially available phosphoramidite. A summary of the sequences used in this study is presented in Table 1. Using recently optimized conditions to minimize DNA degradation and oxidation,76 30 -O-propargyl DNA strands (DNA1or DNA2) were initially clicked to the star polymer. Under these conditions, solutions of DNA and star-polymer in TRIS buffer (pH 7.5) with sodium ascorbate and acetonitrile (ACN) as a minor cosolvent (2%) were purged with argon gas, followed by addition of a Cu2SO4 3 5H2O solution. The use of ACN helps stabilize Cu(I) and prevents DNA degradation due to oxidative damage under aqueous conditions, allowing successful conjugation of DNA (Scheme 2i). This “ligandless” method, using ACN, is in keeping with the philosophy of “click”-chemistry, which simplifies purification, as no additional triazole or other ligand molecules are used in the click conjugation. Following the click reaction, the 2031

dx.doi.org/10.1021/bc200240q |Bioconjugate Chem. 2011, 22, 2030–2037


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Scheme 2. Star Polymer Conjugation to DNA or DNA and Other Functional Molecules Using Click Chemistrya

a The inset depicts the structure at the click linkages. The conjugations were performed using azide functionalized star polymers and 50 -alkyne (i) and 30 -alkyne 2 modifiers on the DNA, respectively. The 30 -alkyne modifiers allow for 50 -end modification of the DNA with useful probes.

Figure 1. Click conjugation permits concomitant loading of molecules along with DNA onto a star polymer. (A) Absorption spectrum of Star/DNA1/ Cy3 conjugate. (B) Fluorescence emission spectra showing FRET between fluorophores conjugated to the star polymer. All emission spectra were taken with an excitation wavelength at 470 nm. The emission spectra of Cy5 (green; on baseline), Cy3 (yellow), and a 1:1 Dy547:Cy5 solution (red) show no FRET between the fluorophores. Once DNA1 (carrying a Dy547 label) and Cy5 were conjugated to the star polymer, the emission spectrum of the conjugate (black) was a result of FRET in the fluorophore pair confirming that the two were attached to the same star polymer.

starDNA conjugates were purified by filtration through a 10 kDa molecular mass cutoff nanosep filter that removes the solvent and ACN as well as unreacted DNA. The conjugation was verified using IR spectroscopy (Supporting Information Figure 1). Complete disappearance of the azide peak at 2200 cm1 suggests that the star-polymers were essentially quantitatively conjugated to the DNA1 and DNA2. Because star-polymers include multiple arms, we sought to demonstrate that click chemistry can be used to incorporate multiple groups onto these arms in a one-step process. Thus, DNA1, that included a 30 -O-propargyl group and 50 -flourescent dye, along with a second fluorescent dye bearing an alkyne were both used for the click reaction with the star polymer (Scheme 2ii).

The two dyes—Dylight547 (Dy547; a Cyanine3(Cy3) equivalent) on the DNA and Cyanine5 (Cy5)—clicked on the starpolymer have known overlapping emission and absorbance regions, respectively, such that, when they are close enough, after excitation of the Dy547, the observed emission is from Cy5 due to F€orster resonance energy transfer (FRET). Following the click reaction and purification of the stars to remove unreacted dyes and DNA, we could observe the absorbance peaks of both dyes (Figure 1A). As the Dy547 dye was at the 50 -terminus of the DNA and the click conjugation was at the 30 -terminus, the presence of the Dy547 absorbance peak indicated that the DNA strand remained intact. Furthermore, we were able to observe FRET emission of the multiconjugate star-DNA1-Cy5 (Figure 1B). 2032



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Figure 2. Controlled DNA self-assembly of star polymers. (A) Scheme for DNA directed self-assembly of star polymers. (B) DLS scans of star polymers click conjugated to DNA1 (black-Ai) and DNA 2 (blue-Aii). DNA directed hybridization of these two stars yield larger assemblies shown in a 1:1 starDNA1/starDNA2 ratio (green-Aiii) and in a 1:10 starDNA1/starDNA2 ratio (purple-Aiv) at 1 nM concentrations.

At the sample dilutions at which the fluorescence was measured, even if the statistically highly improbable reaction outcome produced star-polymer conjugates wherein some stars were only DNA-conjugated while other stars were only dye-conjugated, such a solution would not FRET. Solutions with only one dye or even both dyes in solution together do not result in the FRET emission peak. Thus, the emission due to FRET in the clickconjugate star-polymer solution strongly indicates that both DNA (with dye) and the dye molecule were click-conjugated concomitantly onto the same stars. This shows the power of the click chemistry approach to conjugate DNA as well as other molecules directly onto star-polymers together, taking advantage of the multivalency that the arms provide. DNA-Directed Assembly of Soft Nanoparticle Hybrids. The ability to conjugate DNA by click chemistry onto starpolymers is significant as the functional properties of DNA can be harnessed. While there is a vast array of function that can be derived from DNA, we sought to demonstrate this quite simply by using the hybridization properties of DNA. Using starDNA conjugates, starDNA1 and starDNA2, that include cDNA sequences, we obtained supra-molecular starDNA architectures. DNA directed assembly was accomplished by heating solutions of mixtures of the two starDNA conjugates (at 1 nM) at 95 °C in aqueous buffered salt solution and cooling to room temperature to anneal the DNA strands (Figure 2). We then tested for controlled hybridization behavior by altering the ratios of the starDNA conjugates. Hybridization assembly of the nanoparticles was studied by dynamic light scattering (DLS). As expected, a relationship was found between hybridization ratio and size of the final self-assembled supramolecular architecture. Both starDNA1 and starDNA2 averaged approximately 4 nm. The resulting size of the 1:1 starDNA1/starDNA2 hybrid, Figure 2Aiii, was found to be approximately 9 nm, while a particle size of approximately 20 nm (purple) was observed in the 1:10 starDNA1/starDNA hybrid, Figure 2Aiv, system. No residual free stars were observed in the DLS traces thereby demonstrating the fidelity of cDNA hybridization. These results indicate that self-assembled nanoscale structures can be readily created by using simple cDNA strands conjugated onto star polymers. By simply controlling the ratios of the star DNA hybrids, one can control the size of the resulting particles. Star polymers provide a powerful tool for obtaining these large

assemblies due to the controlled multivalent architecture unavailable to linear DNA systems. Through controlled hybridization, these stars can be used as templates for the design of more advanced DNA architectures. Additionally, nanoparticles with DNA of other functions can be designed and obtained. Reversible Self-Assembly of Soft Nanoparticles using DNA Strand Invasion. The ability to controllably assemble starDNA conjugate particles can be expanded by gaining control over the reverse disassembly process. The ability to control DNA self-assembly using strand invasion techniques has enabled DNA computation.7780 In this scheme, if one of the hybridized strands in a duplex includes a small, single-stranded overhang or “toehold”, the duplex can be invaded by a cDNA strand that includes the toehold region under ambient conditions. This is typically done in linear DNA systems, and we extended this technique to the starDNA hybrids. DNA3 and DNA4, that include partially complementary regions, were conjugated to star polymers, respectively. Additionally, the use of the 50 -phosphohexynyl-terminated DNA, rather than the 30 -O-propargyl DNA, demonstrated how readily the orientation of the conjugated DNA strand can be selected, thereby increasing the informational content loading. These DNA strands were designed such that, upon hybridization assembly of the star-DNA hybrids, a short unhybridized “toehold” region would remain available for an invading strand. DNA5, which is fully complementary to DNA3, can bind to the starDNA3 conjugate displacing starDNA4, in effect, disassembling the particles. This invasion takes place at room temperature and thus allows for detection using DLS. StarDNA3 and StarDNA4 were hybridized (10 nM) at a 1:1 ratio at 95 °C in aqueous buffer (Figure 3; black and red bars). The resulting complex had a mean diameter of 70 nm by DLS (Figure 3B; green bar). This assembly of hybridized starDNA3 to starDNA4 at 10 nM concentrations is large, likely due to the flexibility of the nonbinding region that allows for a larger number of starDNA to bind to one another. In contrast, the starDNA1/starDNA2 complexes that were hybridized at 1 nM concentration and where the strands were completely complementary with no flexible single-stranded region after hybridization formed smaller assemblies. To evaluate control over the assembly, single-stranded DNA5 was used to disassemble the supramolecular structure. 2033



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hybrids with a known number of DNA strands per star, as well as loading arms with DNA and other cargo simultaneously. The control over loading and size distribution is essential for the design and control over self-assembling nanoarchitectures.

’ EXPERIMENTAL SECTION

Figure 3. DNA controlled reversal of star polymerDNA nanoassemblies. (A) Scheme for room-temperature disassembly of nanostructures using an invading DNA strand. (B) Mean area plots of DLS scans indicate a large nanoassembly (green bars) formed using a 1:1 ratio of smaller particles of starDNA3 (black) and starDNA4 (red) at 10 nM concentration can be subsequently gradually disassembled by an invading cDNA strand. The smaller assemblies, depicted in yellow and blue bars, are obtained at 1:100 star polymer/invading DNA and 1:300 star polymer/invading DNA ratios, respectively.

DNA5 was added in 100-fold excess over starDNA3 to the starDNA3/starDNA4 hybrid complex to reduce the supramolecular assemblies from the 70 nm mean size to a hybrid of approximately 10 nm (yellow bar). The excess of invading DNA5 strand was to ensure adequate invasion of the crowded macromolecular complex at ambient temperature. When additional DNA5 was added, up to a 300-fold excess over star-DNA3 (1:300), the system reduced further in size to a mean of 2 nm (blue bar), close to that of the starDNA complexes prior to assembly. Thus, the resulting particle corresponded to a totally disassembled starDNA complex. The slightly larger mean size of the final complex can be attributed to the fact that this starDNA complex is hybridized to DNA5. The phosphate backbone of the DNA caused the arms of the star to extend further due to charge repulsion, while in the parent nonhybridized stars, the single-stranded DNA collapsed into the star arms. Overall, these experiments demonstrate that assembled star DNA hybrids can be selectively invaded. This can lead to starDNA-based detection systems where not only the presence but also the concentration of an invading strand or other stimulus that DNA is sensitive to can be assayed.

’ CONCLUSIONS This paper reports successful DNApolymer conjugation to multifunctional star macromolecules using copper-catalyzed azidealkyne cycloaddition. The application of click chemistry to the growing field of DNApolymer hybrids represents a breakthrough because of the orthogonal control and high yields of this reaction. It is also useful because of ready availability of well-designed azide-terminated polymers, synthesized via CRP methodology.81 DNA-based computing and self-assembly currently relies on either linear DNA with a toehold region80,82 or hybrid macromolecules with an uncontrolled number of DNA arms formed by addition of DNA to nanoparticle templates.8385 This report demonstrates how DNA-based assembly, as well as polymeric systems, can be synergistically expanded in star polymerDNA hybrids. We have developed a procedure that allows for preparation of star copolymers with a controllable number of azide-terminated arms per star. These peripheral functionalized stars can be clicked to alkyne DNA producing

Star Polymer and DNA Synthesis. These were synthesized by standard methods. Details are included in Supporting Information. Click Conjugation of 30 -O-Propargyl DNA and Cy5-Alkyne to Azide Star-Polymers. Stock solutions of CuSO4, sodium ascorbate, and Tris-HCl (pH 7.5) buffer were degassed by bubbling argon through the solutions for 15 min prior to adding DNA. A reaction with 2 μM oligonucleotide in 500 mM Tris (pH = 7.5), 30 mg/mL of star polymer in THF (20 μL THF), 20 mM CuSO4, and 40 mM sodium ascorbate in 3:1 water/ACN. The reaction was run for 2 h with shaking and purified with 10 K nanosep filter (Millipore). The reaction was confirmed by disappearance of the azide stretch at 2260 cm1on IR (Supporting Information Figure 1). In the case of 30 -O-propargyl DNA-Dy547 and concomitant Cy5-alkyne conjugation to a single star polymer, 1.8 μM oligonucleotide and 0.2 μM Cy5-alkyne were introduced into the reaction mixture and the procedure as described above was followed. The resulting purified multivalent conjugate was analyzed using UVvis spectroscopy (NanoDrop 1000) and emission spectroscopy (NanoDrop 3300). FRET controls were done using 100 nM Cy3 (Dy547 equivalent), 100 nM Cy5 solutions. Hybridization Assembly of StarDNA Conjugates. DNAdirected self-assembly of star polymers was achieved by heating solutions of 3 mg/mL (1 nM DNA) starDNA polymer conjugates to 95 °C for 2 min in 100 mM Tris, 1 mM EDTA, and 150 mM NaCl (TEN150 buffer) and cooling to room temperature over 10 min. Following the hybridization cycles, star polymer assembly size was measured using dynamic light scattering (DLS) on a Zetasizer Nano spectrophotometer (Malvern Instruments Ltd.). Controlled Disassembly of StarDNA Nanoassemblies. Strand invasion studies were carried out at a concentration of 10 nM starDNA. After starDNA3 and starDNA4 hybridization, DNA5 was added to a final concentration of 1 μM and 3 μM for a 100- and 300-fold excess over starDNA3, respectively. Remeasurement of solutions following addition of the invading DNA5 strand was carried out after 30 s of incubation at room temperature.

’ ASSOCIATED CONTENT

bS

Supporting Information. Information on chemicals, equipment (DLS, GPC, DNA synthesizer, UVvis, fluorometer, and IR) and the synthesis of star polymers. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*K.M.: Fax (412) 268-6897; Tel (412) 268-3209; E-mail km3b@ andrew.cmu.edu. S.R.D.: Fax (412) 268-1061; Tel (412) 2686871; E-mail [email protected]. Author Contributions #

Authors contributed equally.

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’ ACKNOWLEDGMENT The authors of this paper would like to thank the CRP Consortium and NSF DMR09-69301 for funding. S.R.D. thanks the Department of Chemistry for startup funds. ’ REFERENCES (1) Agut, W., Taton, D., and Lecommandoux, S. (2007) A versatile synthetic approach to polypeptide based rod-coil block copolymers by click chemistry. Macromolecules 40, 5653–5661. (2) Liu, S., Maheshwari, R., and Kiick, K. L. (2009) Polymer-based therapeutics. Macromolecules 42, 3–13. (3) Mei, Y., Beers, K. L., Byrd, H. C. M., Vanderhart, D. L., and Washburn, N. R. (2004) Solid-phase ATRP synthesis of peptidepolymer hybrids. J. Am. Chem. Soc. 126, 3472–3476. (4) Reynhout, I. C., Cornelissen, J. J. L. M., and Nolte, R. J. M. (2009) Synthesis of polymer-biohybrids: from small to giant surfactants. Acc. Chem. Res. 42, 681–692. (5) Borner, H. G. (2009) Strategies exploiting functions and selfassembly properties of bioconjugates for polymer and materials sciences. Prog. Polym. Sci. 34, 811–851. (6) Shakya, A. K., Sami, H., Srivastava, A., and Kumar, A. (2010) Stability of responsive polymer-protein bioconjugates. Prog. Polym. Sci. 35, 459–486. (7) Braunecker, W. A., and Matyjaszewski, K. (2007) Controlled/ living radical polymerization: Features, developments, and perspectives. Prog. Polym. Sci. 32, 93–146. (8) Klok, H. A. (2005) Biological-synthetic hybrid block copolymers: Combining the best from two worlds. J. Polym. Sci., Polym. Chem. 43, 1–17. (9) Le Droumaguet, B., and Nicolas, J. (2010) Recent advances in the design of bioconjugates from controlled/living radical polymerization. Polym. Chem. (U.K.) 1, 563–598. (10) Magnusson, J. P., Saeed, A. O., Fernandez-Trillo, F., and Alexander, C. (2011) Synthetic polymers for biopharmaceutical delivery. Polym. Chem. (U.K.) 2, 48–59. (11) Matyjaszewski, K., and Xia, J. H. (2001) Atom transfer radical polymerization. Chem. Rev. 101, 2921–2990. (12) Wang, J. S., and Matyjaszewski, K. (1995) Controlled living radical polymerization - atom-transfer radical polymerization in the presence of transition-metal complexes. J. Am. Chem. Soc. 117, 5614– 5615. (13) Barbey, R., Lavanant, L., Paripovic, D., Schuwer, N., Sugnaux, C., Tugulu, S., and Klok, H. A. (2009) Polymer brushes via surfaceinitiated controlled radical polymerization: synthesis, characterization, properties, and applications. Chem. Rev. 109, 5437–5527. (14) Bunsow, J., Kelby, T. S., and Huck, W. T. S. (2010) Polymer Brushes: Routes toward Mechanosensitive Surfaces. Acc. Chem. Res. 43, 466–474. (15) Lapienis, G. (2009) Star-shaped polymers having PEO arms. Prog. Polym. Sci. 34, 852–892. (16) Sheiko, S. S., Sumerlin, B. S., and Matyjaszewski, K. (2008) Cylindrical molecular brushes: Synthesis, characterization, and properties. Prog. Polym. Sci. 33, 759–785. (17) Lee, H. I., Pietrasik, J., Sheiko, S. S., and Matyjaszewski, K. (2010) Stimuli-responsive molecular brushes. Prog. Polym. Sci. 35, 24–44. (18) Gao, H., and Matyjaszewski, K. (2009) Synthesis of functional polymers with controlled architecture by CRP of monomers in the presence of cross-linkers: From stars to gels. Prog. Polym. Sci. 34, 317–350. (19) Liu, Y., Dong, Y., Jauw, J., Linman, M. J., and Cheng, Q. (2010) Highly sensitive detection of protein toxins by surface plasmon resonance with biotinylation-based inline atom transfer radical polymerization amplification. Anal. Chem. 82, 3679–3685. (20) Siegwart, D. J., Oh, J. K., Gao, H. F., Bencherif, S. A., Perineau, F., Bohaty, A. K., Hollinger, J. O., and Matyjaszewski, K. (2008) Biotin-,

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