Construction and Characterization of Kilobasepair Densely Labeled

Synthesis and purification of self-assembling peptide-oligonucleotide conjugates by solid-phase peptide fragment condensation. Journal of Peptide Scie...
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Construction and Characterization of Kilobasepair Densely Labeled Peptide-DNA Suzana Kovacic,† Laleh Samii,† Guillaume Lamour,‡ Hongbin Li,‡ Heiner Linke,§ Elizabeth H. C. Bromley,| Derek N. Woolfson,⊥ Paul M. G. Curmi,# and Nancy R. Forde*,† †

Department of Physics, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada § The Nanometer Structure Consortium and Division of Solid State Physics, Lund University, Box 118, 22100 Lund, Sweden | Department of Physics, University of Durham, South Road, Durham, DH1 3L3, United Kingdom ⊥ School of Chemistry and School of Biochemistry, University of Bristol, Bristol, BS8 1TS, United Kingdom # School of Physics, University of New South WalesSydney, New South Wales 2052, Australia ‡

S Supporting Information *

ABSTRACT: Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.

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INTRODUCTION Recent years have seen a rapid development of strategies for creating new biologically compatible materials at the nanoscale. The ability to control, from the bottom-up, the chemical, structural and mechanical features of materials has a broad range of potential applications, including cellular and tissue engineering, biosensing, drug delivery, and control of nanoscale motion.1−7 As one example, the use of nucleic acid hybridization to construct a wide range of DNA origami structures has led to the construction of tailored assemblies at the nanoscale, whose specific functionalization is now seeing widespread use in diverse fields such as photonics, molecular motors, superresolution imaging, structural biology, and chemistry.7−10 For these applications, origami’s nanoscale ordered structure and rigidity are key benefits. Much less developed are modular strategies to functionalize polymers and soft gels at the microscale, for example by modifying kilobasepair-long DNA as a backbone or scaffold. Flexible, porous, larger-scale structures are sought for a variety of applications including drug delivery, tissue engineering, and cell-free translation.1,2 Traditionally made from off-theshelf polymers, recent efforts have targeted their design using sequence-specific building blocks, including proteins,11−16 peptides,2,15,17−19 and DNA.1,20−22 These designed materials form hydrogels, can have well-defined properties and © 2014 American Chemical Society

functionalities (for example, responsive mechanical properties), and have a wide variety of biomedical applications, including tissue engineering.23 Chemical functionalization of these softer materials is not well developed. For peptide scaffolds, challenges center on orthogonality of functional modifications and encoded self-assembly interactions. Given the recent success in creating novel materials by extending the lengths of DNA used in hydrogel scaffolds from tens of basepairs to kilobasepairs,22,24 the ability to modify long DNA structures to introduce complementary functionality, while maintaining the mechanical properties of the scaffold would significantly broaden their possible materials applications. Furthermore, orthogonally addressable functionality in the extended backbone, and its decorated side chains (which could serve as nuclei for intermolecular association) would enable fundamental investigations into the role of tunable cross-link strength and density and their effects on material properties, which for example can be used to guide cell fate in cellular engineering applications.25−27 Other fields such as biosensing and molecular motors design would also benefit from functionalization of extended DNA Received: July 29, 2014 Revised: September 15, 2014 Published: September 18, 2014 4065

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(100 μL, Pierce). The C18 tip was successively washed with 0.1% TFA/5% acetonitrile in water, 10% methanol/0.1% acetic acid in water, and 40% methanol/0.1% acetic acid in water. Azido-PEG4peptide was eluted from the resin with methanol/0.1% acetic acid. The mass of the product was confirmed by electrospray ionization mass spectrometry (ESI-MS; Figure S3). A similar protocol was followed to introduce azide functionalization into the shorter peptide, acetyl-Ser-Asp-Lys-Pro-OH (California Peptide). A total of 1 equiv of the peptide was reacted with 1.3 equiv of NHS-PEG4-azide in DMSO in the presence of N,Ndiisopropylethylamine overnight with rotary mixing. Products were diluted into DMSO, and a preliminary ESI-MS screen was performed to ensure that the reaction had gone to completion. Products were then lyophilized, resuspended in water with a trace of acetonitrile, and added to a C18 gravity column. Elution from the C18 column was performed in 5% increasing step gradients of acetonitrile and water with 0.01% TFA; the majority of purified sample eluted at acetonitrile concentrations of 10 and 15%. The success of the coupling reaction was confirmed by ESI-MS (Figure S4). Preparation and Characterization of Peptide-DNA. To 100 pmol of alkyne-labeled 1 kbp DNA in 10 mM Tris pH 8.5 was added 44 nmol of azido-PEG4-peptide 2 in methanol/0.1% acetic acid or 1 equiv azide to 1 equiv alkyne. The reaction was catalyzed by the addition of copper(II) sulfate (0.5 mM) and ascorbic acid (0.5 mM) in the presence of 0.5 mM TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl] amine) in 47.5% DMSO and 10% methanol (1.3 mL total reaction volume). The reaction was incubated in the dark at room temperature for 2 h with rotary mixing. A control reaction, in which the alkyne-DNA was similarly treated with unmodified peptide 1 was prepared in parallel. After 2 h of incubation, a loose yellow precipitate could be observed in the reaction tube, but not the control tube, suggesting that peptide-DNA 3 falls out of solution as a yellow pellet. Both reaction and control products were purified by overnight ethanol precipitation. Ethanol-precipitated samples were resuspended either in 10 mM Tris, pH 8.5 buffer, or in DMSO. Absorbance spectra were obtained to confirm the presence of peptide and DNA peaks in the peptide-DNA sample and of a DNA peak in the control DNA sample. Peptide-DNA 3 fell out of solution over time but was readily resuspended by gentle pipetting. The insolubility of this construct was further observed in electrophoresis assays (Figure S5). Peptide-DNA 4 was synthesized following the same click chemistry protocol as above for peptide-DNA 3. This product was soluble in aqueous solution and was characterized via agarose gel electrophoresis and atomic force microscopy imaging. Digestion of Peptide-DNA 3 with Trypsin. A total of 5 pmol of peptide-DNA 3 was suspended in 30% DMSO in 10 mM Tris pH 8.5 in the presence of 7.5 μg trypsin (Sigma) in 100 μL of total reaction volume. A total of 5 pmol of unlabeled DNA isolated from the control reaction was similarly treated as a negative control. As positive controls, various concentrations of azido-PEG4-peptide 2 were proteolyzed by trypsin under the same reaction conditions. All reactions were performed at 37 °C. Cleavage of the peptide results in release of the dinitrophenyl quencher; thus, reaction progress was followed by probing for methoxycoumarin emission at 400 nm following excitation into the red edge of its absorbance profile at 360 nm in a fluorescence plate reader (BioTek Synergy). Measurements shown are an average of two replicates. In the presented data, the discontinuity at 20 min was a systematic error among all fluorescent replicates in the plate reader measurement and coincided with operator intervention to change the time scale of kinetic data recording. Atomic Force Microscopy Imaging. Peptide-DNA 4, alkyneDNA, and DNA were deposited on freshly cleaved mica surfaces based on a published protocol.39 Briefly, the samples were diluted to approximately 0.5 nM in 10 mM Tris pH 8.0, 2 mM NaCl, and 2 mM MgCl2. Samples were applied to mica, and after 10 min, NiCl2 at a final concentration of 2.5 mM was added. After a further minute, the mica was rinsed briefly with water and dried with a nitrogen flow. AFM images were obtained in tapping mode on an Asylum Research,

molecules. In the former, the ability to spatially localize large numbers of “bait” molecules for sensing holds promise for signal enhancement.3,28,29 In the latter, introducing repeated toe-holds or substrates for the “feet” of synthetic molecular motors on kbp-long tracks would enable studies of the processivity and performance of these motors to extend from the current nanoscale into microscale distances,4,5,7,30 while taking advantage of DNA as the support structure would avoid challenges of working with alternative approaches such as carbon nanotube functionalization.31 These types of applications can exploit the growing number of modalities available for manipulation and subsequent characterization of single extended DNA molecules.32−35 Previous work functionalizing extended (>1 kbp) stretches of DNA in vitro has incorporated chemically modified deoxyribonucleotide triphosphates (dNTPs) during polymerase chain reaction (PCR) amplification to produce double-stranded DNA decorated with either fluorophores or alkynes.36−38 While one approach utilized directed evolution to produce a DNA polymerase capable of incorporating fluorescently labeled dNTPs,37-38 the other screened commercially available DNA polymerases to find those capable of producing an extended template incorporating the alkyne-modified dNTPs.36 The accessibility of the commercial-based technique, coupled with the widespread use of click chemistry, suggests this as a strategy with broad appeal for modification of extended DNA scaffolds. To do so, it is critical to demonstrate that functionalization of these structures maintains the independence of structure and function. In this work, we demonstrate the synthesis and characterization of kbp-long peptide-DNA hybrid structures, which retain independent peptide and DNA properties. This work enables the possibility of using the complementary, orthogonal structural information on DNA and peptides to expand modular approaches to scaffold and function design in a diverse range of potential applications.

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MATERIALS AND METHODS

Preparation of 1 kbp Alkyne-DNA. A 968 bp (“1 kbp”) DNA product that incorporates alkyne-modified deoxynucleotides was prepared following a literature protocol.30,36 Briefly, PCR amplification of pUC19 plasmid DNA was performed using the following primer pairs: 5′-biotin TTG CTT CGG CCG TTG TAC TGA GAG TGC ACC-3′ (EagI primer), 5′-TGT CTT GGG CCC TTT GGA GCG AAC GAC-3′ (PspOMI primer; Table S1). These primers generate EagI and PspOMI restriction sites at the two ends of the PCR product. To produce modified DNA, the deoxynucleotide dTTP was replaced with the alkyne-modified deoxynucleotide analog C8-alkyne-dUTP ((5-(octa-1,7-diynyl)-2′-deoxyuridine 5′-triphosphate, Jena Bioscience); in control reactions, dTTP was used. DNA was amplified using KOD XL polymerase (Novagen). PCR reaction products were purified using the QiaQuick PCR purification kit (QIAGEN), and their size was confirmed by agarose gel electrophoresis. Preparation of Azido-PEG4-Peptide. One equivalent of the peptide 1, Mca-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2 ((7methoxycoumarin-4-yl)acetyl-L-alanyl-L-prolyl-L-alanyl-L-lysyl-L-phenylalanyl-L-phenylalanyl-L-arginyl-L-leucyl-Nε-(2,4-dinitrophenyl)-L-lysine amide, Peptides International) was reacted with 10 equiv of NHSPEG4-azide (15-azido-4,7,10,13-tetraoxa-pentadecanoic acid succinimidyl ester, Jena Bioscience) in dimethyl sulfoxide (DMSO) in the presence of N,N-diisopropylethylamine (Sigma). The reaction proceeded overnight at room temperature in the dark with rotary mixing. The product was purified by acidifying the reaction mixture to a final concentration of 0.1% trifluoroacetic acid (TFA); then the peptide product was adsorbed onto a pre-equilibrated C18 pipet tip 4066

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Figure 1. Reactions involved in synthesizing extended peptide-DNA hybrid structures. (a) Generation of alkyne-DNA by polymerase chain reaction: dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine triphosphate; dGTP, deoxyguanosine triphosphate; C8-alkyne-dUTP, 5-(octa-1,7diynyl)-2′-deoxyuridine 5′-triphosphate. (b) Synthesis of azido-peptide 2 from unmodified peptide 1 precursor and NHS-PEG4-azide. (c) Functionalization of alkyne-DNA to generate peptide-DNA 3 or 4 via click chemistry. Trypsin cleavage site indicated in red. contour length L, and least-squares fitting to find the persistence length p according to41

Cypher AFM using AC160TS tips with a nominal spring constant of 40 N/m. In the images, alkyne-DNA or peptide-DNA constructs were fitted with parametric splines using custom software.40 Only individual molecules that did not touch or cross were selected for analysis. Height information was extracted from section analyses, which were performed using the AFM software at random locations over a large number of molecules. Persistence lengths were found by determining the mean squared end-to-end distance ⟨R2⟩ as a function of inner

⎛ ⎛ L ⎞⎞⎞ 2p ⎛ ⎜⎜1 − exp⎜ − ⎟⎟⎟⎟⎟ ⟨R2⟩ = 4pL⎜⎜1 − L⎝ ⎝ 2p ⎠⎠⎠ ⎝

(1)

The uncertainty on p was determined using a bootstrapping method: p was calculated for only half of the total number of polymers. The operation was repeated 10 times, and the standard deviation on the 10 values returned for p at each operation gave the uncertainty on p. To 4067

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avoid the underestimation of p that can occur when polymers adsorb before fully equilibrating on the 2D surface, a fractional dimension of 2.5 ± 0.5 was considered.42,43 The assumption of lack of complete 2D equilibration was further supported by analysis of the kurtosis of the θ(L) distribution (where θ is the angle formed by 2 tangents separated by L).41

the DNA (see below), the reaction product had only limited aqueous solubility. Nonetheless, it was amenable to some spectroscopic-based characterization, demonstrating the covalent attachment of peptides to DNA. Absorption spectra show that the peptides were linked successfully to DNA (Figures 2 and S6): absorbance features from 320 to 420 nm are due to the peptide’s methoxycoumarin and dinitrophenyl groups,45 while the strong peak at 260 nm indicates the presence of DNA.

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RESULTS AND DISCUSSION Preparation and Characterization of Alkyne-DNA. PCR amplification of pUC19 plasmid was used to generate a 968 bp (“1 kbp”) alkyne-labeled DNA, in which ∼1/4 of the nucleotide bases displayed an alkyne moiety. This was achieved by completely replacing dTTP with C8-alkyne-dUTP (Figure 1a) during amplification. Both Pwo and KOD XL DNA polymerases36 incorporated C8-alkyne-dUTP into 1 kbp DNA, and using KOD XL polymerase, we were able to generate alkyne-DNA up to ∼3 kbp in length (Supporting Information). As previously observed, relative to control samples alkynelabeled DNA migrated more slowly in agarose gel electrophoresis (Figure S1).36 For use as versatile modular building blocks, the ends of the alkyne-DNA products incorporated restriction sites to facilitate subsequent ligation into diverse DNA platforms. PspOMI and EagI restriction endonucleases were chosen because their recognition sequences lack adenine and thus should remain unmodified following amplification. By designing primers for amplification in which the first alkyne-dUTP was incorporated >5 basepairs from the recognition site (Supporting Information and Figure S2), the resulting products were cleaved by these restriction enzymes and subsequently ligated into longer products.30 These results demonstrate the ability to create, via ligation to orthogonally labeled (or unlabeled) DNA, constructs presenting distinct target sites for interaction and visualization. Preparation and Characterization of Peptide-DNA. From this alkyne-DNA, a peptide-DNA hybrid was prepared, displaying an internally quenched fluorogenic peptide, Mca-AlaPro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2 (1). Trypsin hydrolysis provides an assay for the retention of peptide properties of this commercially available peptide, demonstrating both that the peptide has been linked to the DNA via the lysyl side chain and that the peptide remains accessible to proteolysis. Covalent attachment of the peptide to alkyne-DNA was accomplished first by modifying the ε-amino group of the peptide’s lysyl side chain with NHS-PEG4-azide to introduce the azide moiety necessary for copper-mediated triazole formation (Figure 1b). The PEG spacer was included to reduce potential steric hindrance to dense peptide incorporation along the DNA and to enhance trypsin accessibility to the DNA-linked peptides. Azide modification was confirmed by mass spectrometry (Supporting Information). Azido-peptide 2 was reacted with 1 kbp alkyne-DNA (Figure 1c) at a one-to-one ratio of azido-peptide to alkyne. A reaction between 1 kbp alkyne-DNA and unmodified peptide 1 was performed as a control, and in both reactions, tris(benzyltriazolylmethyl)-amine (TBTA) was added to minimize Cu(I)-induced DNA strand breaks.44 After 2 h at room temperature, a loose yellow precipitate was observed in the reaction sample. Following ethanol precipitation, the product was resuspended in either DMSO or 10 mM Tris, pH 8.5. Due likely to the high local concentration of hydrophobic groups (peptide and dye molecules) arising from their close spacing on

Figure 2. (a) Peptide-DNA hybrid absorbance scan showed the signatures of both DNA and fluorogenic peptide in the product of the click reaction: green trace, peptide-DNA 3; red trace, product of a control reaction between alkyne-DNA and unmodified peptide 1. Enhanced absorbance from 320 to 420 nm in the peptide-DNA sample arose from the peptides’ methoxycoumarin (Mca) and dinitrophenyl (Dnp) groups. (b) Absorption spectra of unreacted peptide remaining in the ethanol supernatant following precipitation, in control and reaction samples. The click reaction used azide-peptide and alkyneDNA, while the control reaction used unmodified peptide with alkyneDNA. These data were analyzed to obtain the estimate of 300 peptides/DNA.

Peptide incorporation efficiency was assessed in two different ways. First, the concentrations of DNA and peptide were estimated from the optical densities, and the ratio of these concentrations was taken. As observed previously, the presence of alkyne groups broadens and slightly shifts the characteristic DNA absorbance peak.36 Values for extinction coefficients used were as follows: 968 bp DNA, ε260 = 1.28 × 107 M−1 cm−1 (assumed to be unchanged from unmodified DNA); methoxycoumarin, ε324 = 12900 M−1 cm−1; dinitrophenyl, ε363 = 15900 M−1 cm−1, ε410 = 7500 M−1 cm−1.45 This method resulted in an estimate of ∼75 peptides/DNA when applied to the construct resuspended in aqueous conditions (Figure 2a). However, this 4068

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value is an underestimate for the following reasons: (i) Dinitrophenyl also contributes to absorbance at 260 nm;45 (ii) Limited solubility of the densely labeled peptide-DNA hybrid could contribute scattering to the optical density at 260 nm; (iii) Control experiments showed that coprecipitated TBTA also contributes to absorbance readings at 260 nm; (iv) If the densely incorporated peptide dyes form aggregates, as has been observed for densely labeled cyanine-DNA,38 a blue-shift in absorbance would occur, contributing further optical density at 260 nm and concomitantly decreasing absorbance in the fluorophore and quencher monomer absorption peaks. For comparison, the same analysis was performed on peptide-DNA resuspended in DMSO (Figure S6). Assuming the same extinction coefficients, this resulted in an estimate of ∼450 peptides/DNA, stoichiometric incorporation of peptide. However, this value is likely to overestimate the labeling density, primarily due to the probable underestimate of DNA concentration in solution due to the strong absorbance by DMSO in the same spectral region. Alternatively, the extent of peptide functionalization of DNA was estimated by comparing the amount of unreacted azidopeptide 2 remaining in the ethanol supernatant following precipitation of peptide-DNA 3 with the control sample (Figure 2b). The difference between the absorbance readings of the two samples corresponds to the amount of azide-modified peptide that reacted with the alkyne-DNA. Using measured absorbances at 324 nm gave an estimate of 264 peptides/DNA, but this value is unreliable because of contributions from absorbance of other soluble species at shorter wavelengths. Measured absorbances attributable to dinitrophenol at two different wavelengths each provided an incorporation density of ∼300 peptides/DNA (292 peptides/DNA at 363 nm; 306 peptides/DNA at 410 nm). This wavelength range has no obvious contributions from other species, and because the absorbance of peptides was measured in free solution, it was not biased by possible spectral shifts arising from dense packing as may occur with the DNA-tethered peptides.38 For these reasons and the self-consistency of results between the two wavelengths, this labeling density was taken as the most reliable estimate and corresponds to a 70% click reaction efficiency (300 of 431 alkyne groups labeled on this 968 bp alkyne-DNA). The resultant peptide density corresponds to approximately one peptide/nm along the DNA backbone. Control over Peptide Density in Peptide-DNA. Peptide density can readily be adjusted by modifying the ratio of azidopeptide to alkyne-DNA during click chemistry coupling. To circumvent problems of aqueous solubility, these experiments used a more hydrophilic peptide (acetyl-Ser-Asp-Lys-Pro-OH) lacking a fluorophore/quencher pair, which resulted in soluble hybrid constructs. Following coupling of the peptide to the DNA as above, purified peptide-DNA 4 was observed by agarose gel electrophoresis (Figure 3). As the ratio of azidopeptide/alkyne-DNA increased, the resulting peptide-DNA band shifted toward higher molecular weight as expected. The monotonic decrease of mobility and the tightness of the band on the gel demonstrate control over the average peptide density and show that the distribution of peptide densities within a given sample is narrow. The ease with which peptide density on DNA can be controlled lends itself well to optimization of labeling density for each desired specific function. Enzymatic Addressability of Peptides in Densely Labeled Peptide-DNA. For incorporation of peptide-DNA

Figure 3. Agarose gel electrophoresis of the 1 kbp peptide-DNA hybrid 4 showed decreasing electrophoretic mobility with an increasing extent of peptide functionalization: lane 1, DNA ladder; lane 2, unlabeled DNA + azido-peptide control; lanes 3−7, alkyneDNA; azido-peptide (4) in increasing relative ratios (significantly less than the absolute molar ratios), as indicated (1×−10×); lane 8, alkyne-DNA + unlabeled peptide control.

hybrid constructs into functional materials or devices, the retention of peptide and DNA properties is essential for independent control over structure and function. To establish whether peptide properties are preserved in densely labeled constructs, the ability of trypsin to hydrolyze peptide-DNA was investigated. Fluorogenic peptide-DNA 3 was used to assess the accessibility of tethered peptides to trypsin, through a kinetic fluorescence assay. Because peptide hydrolysis results in release of the dinitrophenyl quencher from the peptide-DNA construct 3, the fluorescence increase seen following addition of trypsin (Figure 4) indicates successful enzymatic cleavage of tethered peptides. These assays were conducted in a buffer containing 30% DMSO, conditions that maintained trypsin activity (Figure S7), and in which peptide-DNA 3 only very gradually fell out of solution with time (Figure S8). In these conditions, fluorogenic peptide-DNA was hydrolyzed at a slower rate than free peptides, which at comparable concentrations were cleaved almost immediately in this assay (Figure 4). This difference in cleavage rate may arise from the hindered accessibility of bound peptides resulting from reduced solubility of this densely labeled construct. It is likely that a lower density of peptides on the DNA would result in soluble products, since peptides and DNA are each soluble in aqueous conditions; it is at the high local density of peptides in peptide-DNA 3 that insolubility results. This would eliminate the need for DMSO, but here, the choice to work with the densely labeled construct was driven by the desire to obtain sufficiently strong fluorescence signal to clearly establish peptide cleavage. The extent and rate of peptide cleavage from this construct was determined by fitting the time-dependent increase in fluorescence intensity to a single-exponential model: c(t ) = c0(1 − Ae−kclt )

(2)

Here, c(t) represents the time-dependent concentration of cleaved peptides, c0 is its asymptotic limit and kcl is the rate of cleavage. The constant A is included to account for a finite amount of fluorescence signal at t = 0. To relate fluorescence intensity to the amount of peptides cleaved, and to account for the gradual photobleaching of fluorophores, the time at which the peptide-DNA hybrid curve crossed each of the free peptide curves was taken to determine experimental values of c(t). Fitting to these data resulted in a cleavage rate of kcl = 0.027 ± 0.010 min−1 and a concentration of peptides cleaved by trypsin of c0 = 4.0 ± 0.4 μM (Figure 4b). As an alternative approach, photobleaching data of free peptides were fit to a global decay curve, which provided estimates of additional c(t) points for 4069

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Retention of DNA Mobility and Persistence Length in Densely Labeled Peptide-DNA. A further requirement for modular design is the retention of DNA properties in these hybrid constructs. Electrophoretic migration assays (Figure 3) showed that DNA migrates as expected in an agarose gel, with its mobility reduced in a titratable manner with the addition of peptide groups. To examine the effect of covalent modifications on the flexibility of DNA as a polymer, relevant for nanomanipulation and for function as a modular scaffold for hydrogels, its persistence length was determined. AFM imaging was used to assess the height and flexibility of peptide-DNA construct 4 and its precursor alkyne-DNA (Figure 5). Analysis of molecular heights by AFM showed no significant difference between alkyne- and peptide-DNA 4, suggesting that these short tethered peptides do not add enough steric bulk to the construct to be detected by the AFM. The persistence lengths of both alkyne DNA (p = 54 ± 21 nm) and the most densely peptide-labeled DNA 4 (p = 53 ± 20 nm) were found to be consistent with accepted values of unmodified DNA persistence length in standard buffer solutions41,47 and with previous reports on shorter fluorophore-labeled DNA.37 This short peptide is expected to contribute a net negative charge to the hybrid construct, and thus might be expected to increase the persistence length of the DNA through increased electrostatic repulsion. However, the Debye length of 2 nm in the 20 mM ionic strength of the buffer solution used in these experiments is comparable to the length of the PEG4 spacer arm used to link the peptide to the DNA, and thus, it is likely that the unchanged persistence length results from sufficient electrostatic screening by this buffer. Thus, our results show that dense functionalization with alkynes and peptides does not disrupt DNA’s mechanical properties and should permit facile handling, characterization, and assembly with established techniques.

Figure 4. (a) Incubation of peptide-DNA 3 with trypsin resulted in gradually increasing fluorescence from methoxycoumarin (solid red trace), resulting from cleavage of the tethered peptide and release of the dinitrophenyl quencher into solution. Incubation of control DNA with trypsin showed no fluorescence (solid brown trace), as expected. Free azido-peptides, incubated at different concentrations with trypsin, were cleaved almost immediately (dashed lines). To account for photobleaching in solution, from the intersection times of the free peptide cleavage curves with the peptide-DNA curve (arrows), the concentration of peptide cleaved at each time from the hybrid was estimated. (b) Concentration of peptides cleaved from peptide-DNA 3 as a function of time. The red line shows a fit to these data with eq 2, giving a cleavage rate kcl = 0.027 min−1 and saturation at 4.0 μM cleaved peptides.

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CONCLUSIONS In this work, we constructed densely labeled 1 kbp peptideDNA hybrid constructs and demonstrated that they retain peptide and DNA properties. The use of a commercially available DNA polymerase36 to generate peptide-presenting, long DNA substrates suggests the compatibility of this functionalization approach with current methods of DNAbased hydrogel formation.22,24 Moreover, the ease with which these densely functionalized hybrid structures were constructed contrasts with the more stringent demands of alternative approaches for DNA labeling such as origami, where numerous strands must each be labeled in a specific location to present a high local concentration of targets, and where the labeling density is limited to approximately one modification per 6 nm.9 The density of ∼1 peptide/nm achieved here is significantly higher. Because the locations of modifications here are stochastic rather than prescribed as in origami, this represents an average peptide density along the DNA backbone and may be even greater in alkyne-rich regions. There are many potential applications of these extended linear DNA hybrids of tunable peptide density. The use of modular peptide-DNA building blocks, in which the structure and function of each is independently maintained upon incorporation into hybrid structures, suggests the possibility of using both DNA and peptide as orthogonally tunable scaffolds in a given material, enabling the independent driving and tuning of structure and function by both components. The ability to control average peptide density, as shown for peptide-

cleavage of the tethered peptide (Supporting Information and Figure S9). This analysis did not change the asymptotic cleavage (c0 = 4.0 ± 0.2 μM) or rate constant (kcl = 0.023 ± 0.004 min−1). Thus, for a 50 nM hybrid, these estimates corresponded to ∼80 cleaved peptides/peptide-DNA hybrid (∼27% reaction efficiency). This likely underestimates the reaction efficiency for two reasons: (i) the concentration of hybrid is based on the amount of DNA reacted with peptide, which overestimates its final recovered concentration due to small losses during ethanol precipitation and recovery and due to reduced solubility in the cleavage reaction; and, more importantly, (ii) additional quenching of Mca fluorescence may arise from nearby peptide moieties along the DNA backbone, such that 4.0 μM (from comparsion with cleaved peptide in solution) underestimates the concentration of cleaved peptides in the hybrid. Nonetheless, this assay demonstrated that a significant portion of tethered peptides remains accessible to protease recognition and cleavage. They can thus be considered as addressable entities when incorporated along the DNA backbone, where they could serve for example as substrates for designed proteolytic molecular motors.7,46 4070

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Figure 5. AFM images and analyses of alkyne-DNA and peptide-DNA 4. (a) Sample AFM image of 1 kbp alkyne-DNA. Scale bar = 100 nm for (a) and (d). (b) Distribution of heights measured for alkyne-DNA. (c) Determination of persistence length of p = 54 ± 21 nm for alkyne-DNA (N = 178 molecules), using eq 1. (d) Sample AFM image of 1 kbp peptide-DNA construct 4, at highest labeling density (lane 7, Figure 3). (e) Distribution of heights measured for peptide-DNA 4. (f) Determination of persistence length of p = 53 ± 20 nm for peptide-DNA 4 (N = 130).

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DNA 4 in this work, may prove particularly useful when tuning properties such as hydrogel cross-link density and backbone solubility at high concentrations. To enhance their versatility, these constructs could be further incorporated into more complex structures through ligation to orthogonally labeled DNA, presenting diblock or multiblock polymeric systems.30,48 The use of DNA as a one-dimensional backbone for assembly has applications in synthetic molecular motor design,4−7,30,31,46 where its presented cleavable peptides could be used to direct biased motion.7,31,46 The advantages of this approach over alternative linear templating strategies31 include the growing variety of techniques available for the nanomanipulation of DNA,32−35 which should be directly applicable to peptide-DNA due to its preserved persistence length. The ability to controllably and densely present peptides on DNA may also prove fruitful for biosensing, cell capture and drug delivery, where presentation of a high local concentration of targets can strongly enhance signal through polyvalency and cooperativity.3,28,29,49 In summary, the ability to control in a facile manner the density of peptide modifications on DNA has the potential to open the door to a wide variety of applications in nanoscale device design, biosensing, and responsive materials development.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a research grant from the Human Frontier Science Program (Grant No. RGP31/2007), by NSERC Discovery Grants (Forde, Li), and the Swedish Research Council (Linke). We thank Andrew Wieczorek for useful discussions and Dr. Hongwen Chen for conducting the ESI-MS in the laboratory of Dr. Robert Young.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Supporting text, Tables S1 and S2, and Figures S1−S9. This material is available free of charge via the Internet at http:// pubs.acs.org. 4071

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Biomacromolecules

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