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A Protein−Polymer Hybrid Mediated By DNA Saadyah E. Averick,† Eduardo Paredes,†,‡ Debasish Grahacharya,†,‡ Bradley F. Woodman,§ Shigeki J. Miyake-Stoner,§ Ryan A. Mehl,§ 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 § Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604, United States ABSTRACT: Protein−polymer hybrids (PPHs) represent an important and rapidly expanding class of biomaterials. Typically in these hybrids the linkage between the protein and the polymer is covalent. Here we describe a straightforward approach to a noncovalent PPH that is mediated by DNA. Although noncovalent, the DNA-mediated approach affords the highly specific pairing and assembly properties of DNA. To obtain the protein−DNA conjugate for assembly of the PPH, we report here the first direct copper catalyzed azide−alkyne cycloaddition-based protein−DNA conjugation. This significantly simplifies access to protein−DNA conjugates. The protein−DNA conjugate and partner polymer−DNA conjugate are readily assembled through annealing of the cDNA strands to obtain the PPH, the assembly of which was confirmed via dynamic light scattering and fluorescence spectroscopy. prosthetic groups,38,39 biotin−avidin interactions,40−43 modification of native amino acids (e.g., coupling to lysine or cysteine)44−47 or the enzymatic modification of proteins to introduce appropriate orthogonal conjugation sites.48,49 In these methods DNA is conjugated to a protein of interest via labile disulfide linkages,50 apo-enzymes and their prosthetic groups (such as a heme),51,38,39 or additional fusion proteins that covalently link to molecular tags (such as the HaloTag conjugate).52 Alternately, maleimide or N-hydroxy-succinimide (NHS) ester and related chemistries requiring nucleophilic displacement are used.33 Still, there exists considerable scope for improving reaction efficiencies and purification protocols toward more facile access to DNA−protein conjugates. Click-chemistry, with its high efficiency in aqueous media for nucleic acid modifications,53−55 among other advantages, has only recently been used for protein−DNA conjugates. Distefano and co-workers enzymatically and site-specifically labeled a protein with an azido modified isoprenoid that could be used for click-conjugation to an alkyne-functionalized DNA.48 In this work we demonstrate a direct approach to synthesizing a DNA-mediated PPH by direct conjugation of DNA, via click-chemistry, to a genetically engineered azido functional protein and an azido functional polymer, respectively.

1. INTRODUCTION Protein−polymer hybrids (PPHs) are an important class of biomaterials with a wide range of applications for biotechnology, including sensors1,2 and therapeutics.3−5 These materials are typically prepared by “grafting from” or “grafting to” methods.6−9 In the “grafting from” method, an initiating group is immobilized on the protein, and the polymer is grown in situ.10−14 In the “grafting to” method, the most prevalent method of PPH preparation, preformed polymers are conjugated to proteins.15,16 New paradigms in the field of PPHs have emerged through the ability to genetically engineer proteins with predetermined stoichiometry, site-specificity, and even unnatural amino acids (UAAs).17−23 Using genetic engineering approaches, poly(ethylene glycol) (PEG) has been successfully attached to proteins using copper-catalyzed azide−alkyne cycloaddition (“click-chemistry”)24 or ketoneamine-oxy chemistry.25 Recently, click-chemistry has been applied to the synthesis of polymer−DNA conjugates26,27 with DNA mediation of the reversible assembly of the starpolymer soft nanoparticles.26 If direct DNA conjugates on polymer chains could be likewise obtained, they would provide upfront access to DNA-mediated PPH through assembly with partner protein−DNA conjugates. The use of DNA that provides exceptional control through specific hybridization has tremendously propelled the fields of biotechnology and nanotechnology forward.28−34 The conjugation of DNA with proteins has led to new methods in the preparation of sensing devices and nanostructured objects.35,36,33 Since Niemeyer’s pivotal demonstration of supramolecular assembly through biotinylated DNA and streptavidin binding,37 there have been several elegant approaches to DNA−protein conjugation and its applications. These approaches have used © 2012 American Chemical Society

Special Issue: Bioinspired Assemblies and Interfaces Received: October 18, 2011 Revised: December 9, 2011 Published: January 6, 2012 1954

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Figure 1. Synthesis of DNA−protein and DNA−polymer conjugates required for PPH assembly. (A) Schematic representing individual components of protein, DNA, and polymer used for click conjugations. Both protein and polymer include an azide, and the DNA sequences (DNA1: 5′-GCG TTT GCT CTT CTT GCG and cDNA1′: 5′-Dy547-CGC AAG AAG AGC AAA CGC) include a 3′-terminal ribose residue with alkyne for direct click conjugation (B) to furnish GFP-t-DNA1 and pOEOMA-t-DNA1′. Inset shows the resulting triazole linkage. (C) UV−vis characterization of conjugates: (i) absorption spectra of 10 μM solutions of GFP-N3 before (blue line) and after (red line) DNA1 conjugation to form GFP-t-DNA1 in 1X PBS buffer; inset is detail of GFP absorption peak that is unaffected by the click-conjugation; (ii) absorption spectra of a 0.1 mg/mL solution of N3-pOEOMA in 1X PBS buffer before (blue line) and after (red line) DNA1′ conjugation to form pOEOMA-t-DNA1′; inset shows region of Dy547 absorption peak.

2. EXPERIMENTAL SECTION Commercially available compounds were used without further purification unless otherwise noted. CuBr 2 (98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDTA, 98%), N,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), 2-bromo-2-phenylacetic acid, 2-(2-(2chloroethoxy)ethoxy)ethanol, 4,4′-dinonyl-2,2′-bipyridine (dNBpy), and toluene were purchased from Aldrich. CuBr (98%, Acros) was purified by stirring in acetic acid, filtered, washed with 2-propanol, and then dried under vacuum. Oligo(ethylene oxide) monomethyl ether methacrylate (average molecular weight ∼154 and 300, OEO 2 MA and OEOMA300) was purchased from Aldrich and purified by passing through a column filled with basic alumina to remove the inhibitor and/or antioxidant. Phosphoramidites with labile phenoxyacetyl (PAC) protecting groups and appropriate reagents for solid phase synthesis of DNA were purchased from ChemGenes or Glen Research, and the controlled pore glass (CPG) columns with 3′-O-propargyl residues for the DNA synthesis were purchased from ChemGenes. The Dylight 547 (Dy547) phosphoramidites were purchased from Glen Research. Copper sulfate pentahydrate (CuSO4·5H2O) was purchased from Sigma Aldrich. HPLC-grade acetonitrile (ACN) was purchased from Fisher. Sodium ascorbate was purchased from Alfa Aesar. Other solvents and reagents not otherwise specified were purchased from Fisher. Molecular weight and polydispersity were measured by gel permeation chromatography (GPC; Polymer Standards Services-PSS) columns (guard, 105, 103, and 102 Å), with tetrahydrofuran (THF) eluent at 35 °C, flow rate of 1.00 mL/min, and differential refractive index (RI) detector (Waters, 2410). Toluene was used as the internal standard to correct for any fluctuation of the THF flow rate. The apparent molecular weights and polydispersity were determined with a calibration based on linear poly(methyl methacrylate) standards

using WinGPC 6.0 software from PSS. Dynamic light scattering (DLS) was conducted on a Malvern Zetasizer using 40 μL disposable cuvettes. UV−vis spectra were obtained on a NanoDrop 1000 spectrophotometer. Emission spectra were obtained on a TECAN sapphire plate reader. Expression and Purification of Green Fluorescent Protein (GFP)-Azide. DH10B Escherichia coli cells cotransformed with one pBad vector and pDule1-pCNF. The pDule1pCNF plasmid has high fidelity and efficiency for site specifically incorporation of a variety of UAAs including pN3F(1). The pBad-sf GFP-134TAG vectors were used for producing GFP-135pN3F. The cotransformed cells were used to inoculate 5 mL of noninducing medium containing 100 μg/ mL Amp and 25 μg/mL Tet. The noninducing medium culture was grown to saturation with shaking at 37 °C, and 5.0 mL was used to inoculate 0.5 L autoinduction medium with 100 μg/mL Amp, 25 μg/mL Tet, and 1 mM pN3F (0.5 L of media grown in 2 L plastic baffled flasks). After 40 h of shaking at 37 °C, cells were collected by centrifugation. The protein was purified using BD-TALON cobalt ionexchange chromatography. The cell pellet was resuspended in wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, pH 7) containing 1 mg/mL chicken egg white lysozyme, and sonicated 3 × 1 min while cooled on ice. The lysate was clarified by centrifugation, applied to 6−9 mL bedvolume resin, and bound for 30 min. Bound resin was washed with >50 volumes wash buffer. Protein was eluted from the bound resin with 2.5 mL aliquots of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole pH 7) until the resin turned pink and the color of the eluent the column was no longer green. The eluents concentrations were determined with a Bradford protein assay. The protein fractions were desalted into phosphate buffered saline (PBS) using PD10 columns and concentrated with 3000 MWCO centrifuge filters. 1955

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DNA1′ with pOEOMA. Forty microliters of pOEOMA (100 μM) was combined with 50 μL DNA1′ (676 μM) and 10 μL of sodium ascorbate stock in a 400 μL Eppendorf vial and degassed by bubbling with argon for 5 min. To this solution degassed CuSO4·5 H2O+PMDTA stock was added, and the reaction was vortexed for 2 h and purified with 30 K AmiconUltra (Millipore) centrifuge filter to obtain pOEOMA-t-DNA1′ that was stored in 1X PBS buffer. Hybridization. A 20 μL solution of GFP-t-DNA1 (10 μM in 1X PBS buffer) was combined with 20 μL of pOEOMA-tDNA1′ (10 μM in 1X PBS buffer). The solution was heated to 65 °C for 3 min and then cooled to room temperature.

Components for autoinducing and noninducing media, for final volume of 500 mL: Autoinducing media: 5% aspartate, pH 7.5 (25 mL), 10% glycerol (25 mL), 25X 18 amino acid mix (20 mL), 50X M (10 mL), leucine (4 mg/mL), pH 7.5 (5 mL), 20% arabinose (1.25 mL), 1 M MgSO4 (1 mL), 40% glucose (625 μL), trace metals (100 μL). Noninducing medium: 5% aspartate, pH 7.5 (25 mL), 25X 18 amino acid mix (20 mL), 50X M (10 mL), leucine (4 mg/mL), pH 7.5 (5 mL), 1 M MgSO4 (1 mL), 40% glucose (6.25 mL), trace metals (100 μL). DNA Synthesis. Solid phase oligonucleotide synthesis was performed on a MerMade 4 instrument (Bioautomation). Synthesis of the oligonucleotides was conducted on commercially available solid support columns and performed with standard commercially available phosphoramidites as directed by the manufacturer. Cleavage off the solid support and base deprotection of the oligonucleotides was performed by using ammonium hydroxide at 65 °C for 2 h and standard protocols for deprotection as recommended by the manufacturer. Desalting and purification was conducted using C18 columns (Waters) and protocols recommended by the manufacturer. The strands synthesized are as described in Figure 1. Polymer Synthesis. Azido ATRP initiator synthesis: (N3− PEG3−BPA) (BPA = bisphenol A): In a 250 mL round-bottom flask (rbf) 2-(2-(2-azidoethoxy)ethoxy)ethanol (2.00 g, 11.4 mmol) was dissolved in 100 mL of dichloromethane. To this solution DCC (2.59 g, 14.9 mmol) and DMAP (0.139 g, 1.1 mmol) were added. 2-Bromo-2-phenylacetic acid was added portion-wise, and the reaction was stirred at room temperature under nitrogen for 16 h. The reaction mixture was extracted with water (2X 50 mL), 0.1 N NaOH (2X 50 mL) and once with brine (50 mL). The organic layer was dried using sodium sulfate filtered and evaporated under reduced atmosphere. N3− PEG3−OH:2-bromo-2-phenyl acetic acid:DCC:DMAP[1:1.3:1.1:0.1] MZ: 395 (M+Na+) 300 MHz 1H NMR (CDCl3): 3.476 (2H, t), 3.849−3.732 (8H, m), 5.517 (1H, s), 7.69−7.448 (5H, m). Azido-pOEOMA. To a 10 mL Schlenk flask N3−PEG3− BPA (30 mg, 0.09 mmol), OEOMA300 (0.7549 mL, 2.62 mmol), OEOMA2(1.126 mL, 6.11 mmol), CuBr2 (1.9 mg, 0.01 mmol), dNBpy (38.2 mg, 0.18 mmol) and 2 mL of toluene were added. The flask was sealed and bubbled for 20 min with nitrogen. The flask was then frozen, and under the flow of nitrogen CuBr (11.3 mg, 0.08 mmol) was added. The flask was backfilled with nitrogen and heated at 60 °C for 2 h. The reaction was quenched by adding 15 mL of THF. The reaction mixture was passed through a column of neutral alumina, concentrated, and precipitated into 50 mL of ethyl ether. Mn = 20 000; Mw/Mn = 1.42. The pOEOMA solution has a lower critical solution temperature (LCST) of 39 °C. DNA Conjugations. Stock solutions of CuSO4·5H2O precomplexed with PMDTA (10 μM CuSO4 and 50 μM PMDTA), sodium ascorbate (50 μM), and Tris-HCl (pH 7.5) buffer were degassed by bubbling argon through the solutions for 15 min prior to adding DNA. DNA1 with GFP-N3. Forty microliters of GFP-N3 (100 μM) was combined with 40 μL of DNA1 (800 μM) and 10 μL of sodium ascorbate stock in a 400 μL Eppendorf vial and degassed by bubbling with argon for 5 min. To this solution degassed CuSO4·5H2O+PMDTA stock was added, and the reaction was vortexed for 2 h and purified with a 30 K AmiconUltra (Millipore) centrifuge filter to obtain GFP-tDNA1 (t = resultant triazole) that was stored in 1X PBS buffer.

3. RESULTS AND DISCUSSION Here we sought a direct approach to protein−DNA conjugates, leveraging advances in protein engineering, to obtain a protein bearing an azide for use in click chemistry conjugation with DNA. We therefore expressed green fluorescent protein (GFP) containing an azide functionality through UAA incorporation at a single, surface-exposed residue. An aminoacyl-tRNA synthetase/tRNA pair was used for the cotranslational and site-specific incorporation of p-azidophenylalanine (pN3F) into proteins.56,57 This single set of genetic machinery permitted the incorporation of pN3F into GFP. The resultant genetically engineered GFP with pN3F at its 134th amino acid residue (GFP-N3) was directly click-conjugated in aqueous buffer to synthetic DNA1 that included a 3′-terminal alkyne (Figure 1A,B), to yield GFP-t-DNA1 (t indicates the resultant triazole linkage). The benign reaction conditions can be readily evidenced by the unaltered absorbance properties of the GFP in the conjugate (Figure 1C(i)). Given that the protein was folded and purified after expression, following the clickconjugation, no additional purification besides a simple centrifugal filtration was needed. While the individual techniques of UAA incorporation and click-chemistry are well-known, our direct approach expands the range of methods and appreciably simplifies demonstrated access to protein− DNA conjugates. To obtain a polymer with a reactive handle for DNA conjugation, we prepared an azido functional polymer (N3pOEOMA, Mn = 20 000, Mw/Mn = 1.42 (Figure 1A)) using atom transfer radical polymerization (ATRP).32,58−66 A second DNA oligomer, DNA1′ (complementary in sequence to DNA1) that includes both a 3′-terminal alkyne and a 5′fluorescent dye (Dy547, a Cy3 equivalent) was synthesized. The 3′-terminal alkyne of DNA1′ was click-conjugated to N3pOEOMA under mild aqueous conditions (Figure 1A,B), to yield pOEOMA-t-DNA1′. Removal of unreacted DNA and catalyst was accomplished by using Amicon ultracentrifugal filters (30 kDa molecular weight cutoff (MWCO)). The absorption spectrum of the polymer following conjugation to the DNA shows a clear peak at 547 nm that corresponds to the dye attached to the DNA (Figure 1C(ii)) Next we sought to obtain a PPH with GFP-t-DNA1 and pOEOMA-t-DNA1′ by hybridizing the cDNA strands. Solutions of GFP-t-DNA1 and pOEOMA-t-DNA1′ each at a 2 μM concentration were simply mixed together or mixed and then annealed by heating to 65 °C for 3 min followed by cooling to room temperature (Figure 2A) The resulting solutions of conjugates as simply mixed or annealed hybrids were analyzed using DLS and fluorescence (Figure 2B,C). The hydrodynamic diameter (D) was determined by volume distributions using DLS at 30 °C (Figure 2B). For GFP-t1956

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heating and cooling the aqueous buffer solutions. The resulting hybrid is the first example of a noncovalent DNA-mediated PPH. The formation of the hybridized PPH was confirmed by DLS that showed a clear increase of the hybrid size compared to the starting materials. Further, the hybrid displayed FRET between GFP and the Dy547 fluorescent dye on the DNA of the hybrid partner. FRET was not observed in the unhybridized starting materials as simply mixed, but was detected after the heating and cooling annealing steps required for hybridization. The FRET between GFP and Dy547 resulting from DNA hybridization in essence shows the uniting of a polymer and protein in a novel approach to PPH. The noncovalent DNA mediation approach presented in this paper is currently being used toward the creation of smart PPHs that take advantage of the informational and functional properties of DNA as well as the high tailorability of polymers. We envision that the methods disclosed in this manuscript can be applied to prepare a wide range of DNA-mediated functionalized polymer, supramolecular, and advanced hybrid materials and surfaces.

Figure 2. (A) DNA-mediated PPHs. (B) Analysis of hybridization between GFP-t-DNA1 and pOEOMA-t-DNA1′ using DLS at 0.1 mg/ mL 30 °C. GFP-t-DNA1 (D = 3.8 nm CV = 0.38, black line), pOEOMA-t-DNA1′ (D = 6.4 nm CV= 0.26, red line), and GFP-tDNA1 and pOEOMA-t-DNA1′ (D = 26.6 nm CV= 0.25, hybridized, blue line). (C) Analysis of hybridization-mediated FRET GFP-t-DNA1 and pOEOMA-t-DNA1′ in 1X PBS buffer; excitation 480 nm, emission 505 nm at 25 °C. Fluorescent emission of 1 μM samples of GFPDNA1 (black line), GFP-t-DNA1 and pOEOMA-t-DNA1′ (mixed, red line), and GFP-t-DNA1 and pOEOMA-t-DNA1′ (hybridized, blue line).

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AUTHOR INFORMATION

Corresponding Author

*Fax: 412-268-6897; tel: 412-268-3209; e-mail: km3b@andrew. cmu.edu (K.M.). Fax: 412-268-1061; tel: 412-268-6871; e-mail: [email protected] (S.R.D.).

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ACKNOWLEDGMENTS The authors of this paper would like to thank the CRP Consortium and NSF DMR 09-69301 for funding. S.R.D. thanks the Department of Chemistry for startup funds. NMR instrumentation at CMU was partially supported by the NSF (CHE-0130903 and CHE-1039870).

DNA1, we could determine D = 3.8 nm (coefficient of variance, CV = 0.38, black line, Figure 2B) and for pOEOMA-t-DNA1′ D = 6.4 nm (CV = 0.26, red line, Figure 2B). A shift to a larger size of D = 26.6 nm with a CV = 0.25 was readily observed for the GFP-t-DNA1/pOEOMA-t-DNA1′ hybrid (blue line, Figure 2B). Whereas the DNA strands in the component conjugates are single stranded, hybridization would result in a more rigid duplex, thereby the clear increase in size after hybridization demonstrates the successful preparation of a DNA-mediated PPH. To analyze the formation of the hybrid further, fluorescence spectroscopy was used (Figure 2C). The absorbance of Dy547 overlaps with the emission of GFP, resulting in Förster resonance energy transfer (FRET), if the two fluorophores are in close proximity. Thus, fluorescence spectroscopy allows for confirmation of successful hybridization. The simple mixture of GFP-t-DNA1 and pOEOMA-t-DNA1′ (red line, Figure 2C) could be clearly distinguished from the hybrid GFPt-DNA1/pOEOMA-t-DNA1′ (blue line, Figure 2C). In the GFP-t-DNA1 mixture with pOEOMA-t-DNA1′, no FRET is observed with the emission spectrum essentially overlapping with the GFP-t-DNA1 (black line, Figure 2C), suggesting that when mixed simply, the two conjugates remain distinct. By contrast, the emission spectrum of the annealed and thereby hybridized GFP-t-DNA1/pOEOMA-t-DNA1′ displays a clear peak at 570 nm (emission of Dy547) with reduced emission at 508 nm (emission of GFP) indicative of FRET between GFP and Dy547 that can only result following specific DNA hybridization. Thus fluorescence spectroscopy clearly confirms the DNA-mediated hybridization to form a PPH.

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REFERENCES

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dx.doi.org/10.1021/la204077v | Langmuir 2012, 28, 1954−1958