construction and characterization of kilobasepair densely

Construction and Characterization of Kilobasepair Densely LabeledPeptide-DNASuzana 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 WalesSydney, New South Wales 2052, Australia

*S Supporting Information

ABSTRACT: Directed assembly of biocompatible materials benefits frommodular building blocks in which structural organization is independent ofintroduced functional modifications. For soft materials, such modificationshave been limited. Here, long DNA is successfully functionalized with densedecoration by peptides. Following introduction of alkyne-modified nucleo-tides into kilobasepair DNA, measurements of persistence length show thatDNA mechanics are unaltered by the dense incorporation of alkynes (∼1alkyne/2 bp) and after click-chemistry attachment of a tunable density ofpeptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp)demonstrates addressability of the functional groups, showing that thisaccessible 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 futureapplications, such as building blocks in modular approaches to a range of problems in synthetic biology.

■ INTRODUCTIONRecent years have seen a rapid development of strategies forcreating 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 broadrange of potential applications, including cellular and tissueengineering, biosensing, drug delivery, and control of nanoscalemotion.1−7 As one example, the use of nucleic acid hybrid-ization to construct a wide range of DNA origami structures hasled to the construction of tailored assemblies at the nanoscale,whose specific functionalization is now seeing widespread usein diverse fields such as photonics, molecular motors, super-resolution imaging, structural biology, and chemistry.7−10 Forthese applications, origami’s nanoscale ordered structure andrigidity are key benefits. Much less developed are modularstrategies to functionalize polymers and soft gels at themicroscale, for example by modifying kilobasepair-long DNAas a backbone or scaffold.Flexible, porous, larger-scale structures are sought for a

variety of applications including drug delivery, tissue engineer-ing, and cell-free translation.1,2 Traditionally made from off-the-shelf polymers, recent efforts have targeted their design usingsequence-specific building blocks, including proteins,11−16

peptides,2,15,17−19 and DNA.1,20−22 These designed materialsform hydrogels, can have well-defined properties and

functionalities (for example, responsive mechanical properties),and have a wide variety of biomedical applications, includingtissue engineering.23 Chemical functionalization of these softermaterials is not well developed. For peptide scaffolds,challenges center on orthogonality of functional modificationsand encoded self-assembly interactions. Given the recentsuccess in creating novel materials by extending the lengthsof DNA used in hydrogel scaffolds from tens of basepairs tokilobasepairs,22,24 the ability to modify long DNA structures tointroduce complementary functionality, while maintaining themechanical properties of the scaffold would significantlybroaden their possible materials applications. Furthermore,orthogonally addressable functionality in the extended back-bone, and its decorated side chains (which could serve as nucleifor intermolecular association) would enable fundamentalinvestigations into the role of tunable cross-link strength anddensity and their effects on material properties, which forexample can be used to guide cell fate in cellular engineeringapplications.25−27

Other fields such as biosensing and molecular motors designwould also benefit from functionalization of extended DNA

Received: July 29, 2014Revised: September 15, 2014Published: September 18, 2014

Article

pubs.acs.org/Biomac

© 2014 American Chemical Society 4065 dx.doi.org/10.1021/bm501109p | Biomacromolecules 2014, 15, 4065−4072

pubs.acs.org/Biomac

molecules. In the former, the ability to spatially localize largenumbers of “bait” molecules for sensing holds promise forsignal enhancement.3,28,29 In the latter, introducing repeatedtoe-holds or substrates for the “feet” of synthetic molecularmotors on kbp-long tracks would enable studies of theprocessivity and performance of these motors to extend fromthe current nanoscale into microscale distances,4,5,7,30 whiletaking advantage of DNA as the support structure would avoidchallenges of working with alternative approaches such ascarbon nanotube functionalization.31 These types of applica-tions can exploit the growing number of modalities available formanipulation and subsequent characterization of singleextended DNA molecules.32−35

Previous work functionalizing extended (>1 kbp) stretches ofDNA in vitro has incorporated chemically modified deoxy-ribonucleotide triphosphates (dNTPs) during polymerase chainreaction (PCR) amplification to produce double-stranded DNAdecorated with either fluorophores or alkynes.36−38 While oneapproach utilized directed evolution to produce a DNApolymerase capable of incorporating fluorescently labeleddNTPs,37-38 the other screened commercially available DNApolymerases to find those capable of producing an extendedtemplate incorporating the alkyne-modified dNTPs.36 Theaccessibility of the commercial-based technique, coupled withthe widespread use of click chemistry, suggests this as a strategywith broad appeal for modification of extended DNA scaffolds.To do so, it is critical to demonstrate that functionalization ofthese structures maintains the independence of structure andfunction.In this work, we demonstrate the synthesis and character-

ization of kbp-long peptide-DNA hybrid structures, whichretain independent peptide and DNA properties. This workenables the possibility of using the complementary, orthogonalstructural information on DNA and peptides to expandmodular approaches to scaffold and function design in adiverse range of potential applications.

■ MATERIALS AND METHODSPreparation of 1 kbp Alkyne-DNA. A 968 bp (“1 kbp”) DNA

product that incorporates alkyne-modified deoxynucleotides wasprepared following a literature protocol.30,36 Briefly, PCR amplificationof pUC19 plasmid DNA was performed using the following primerpairs: 5′-biotin TTG CTT CGG CCG TTG TAC TGA GAG TGCACC-3′ (EagI primer), 5′-TGT CTT GGG CCC TTT GGA GCGAAC GAC-3′ (PspOMI primer; Table S1). These primers generateEagI and PspOMI restriction sites at the two ends of the PCR product.To produce modified DNA, the deoxynucleotide dTTP was replacedwith the alkyne-modified deoxynucleotide analog C8-alkyne-dUTP((5-(octa-1,7-diynyl)-2′-deoxyuridine 5′-triphosphate, Jena Bio-science); in control reactions, dTTP was used. DNA was amplifiedusing KOD XL polymerase (Novagen). PCR reaction products werepurified using the QiaQuick PCR purification kit (QIAGEN), andtheir 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 ((7-methoxycoumarin-4-yl)acetyl-L-alanyl-L-prolyl-L-alanyl-L-lysyl-L-phe-nylalanyl-L-phenylalanyl-L-arginyl-L-leucyl-Nε-(2,4-dinitrophenyl)-L-ly-sine amide, Peptides International) was reacted with 10 equiv of NHS-PEG4-azide (15-azido-4,7,10,13-tetraoxa-pentadecanoic acid succini-midyl ester, Jena Bioscience) in dimethyl sulfoxide (DMSO) in thepresence of N,N-diisopropylethylamine (Sigma). The reactionproceeded overnight at room temperature in the dark with rotarymixing. The product was purified by acidifying the reaction mixture toa final concentration of 0.1% trifluoroacetic acid (TFA); then thepeptide product was adsorbed onto a pre-equilibrated C18 pipet tip

(100 μL, Pierce). The C18 tip was successively washed with 0.1%TFA/5% acetonitrile in water, 10% methanol/0.1% acetic acid inwater, and 40% methanol/0.1% acetic acid in water. Azido-PEG4-peptide was eluted from the resin with methanol/0.1% acetic acid. Themass of the product was confirmed by electrospray ionization massspectrometry (ESI-MS; Figure S3).

A similar protocol was followed to introduce azide functionalizationinto the shorter peptide, acetyl-Ser-Asp-Lys-Pro-OH (CaliforniaPeptide). A total of 1 equiv of the peptide was reacted with 1.3equiv of NHS-PEG4-azide in DMSO in the presence of N,N-diisopropylethylamine overnight with rotary mixing. Products werediluted into DMSO, and a preliminary ESI-MS screen was performedto ensure that the reaction had gone to completion. Products werethen lyophilized, resuspended in water with a trace of acetonitrile, andadded to a C18 gravity column. Elution from the C18 column wasperformed in 5% increasing step gradients of acetonitrile and waterwith 0.01% TFA; the majority of purified sample eluted at acetonitrileconcentrations of 10 and 15%. The success of the coupling reactionwas confirmed by ESI-MS (Figure S4).

Preparation and Characterization of Peptide-DNA. To 100pmol of alkyne-labeled 1 kbp DNA in 10 mM Tris pH 8.5 was added44 nmol of azido-PEG4-peptide 2 in methanol/0.1% acetic acid or 1equiv azide to 1 equiv alkyne. The reaction was catalyzed by theaddition of copper(II) sulfate (0.5 mM) and ascorbic acid (0.5 mM) inthe presence of 0.5 mM TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine) in 47.5% DMSO and 10% methanol (1.3 mL totalreaction volume). The reaction was incubated in the dark at roomtemperature for 2 h with rotary mixing. A control reaction, in whichthe alkyne-DNA was similarly treated with unmodified peptide 1 wasprepared in parallel. After 2 h of incubation, a loose yellow precipitatecould 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 ethanolprecipitation. Ethanol-precipitated samples were resuspended either in10 mM Tris, pH 8.5 buffer, or in DMSO. Absorbance spectra wereobtained to confirm the presence of peptide and DNA peaks in thepeptide-DNA sample and of a DNA peak in the control DNA sample.Peptide-DNA 3 fell out of solution over time but was readilyresuspended by gentle pipetting. The insolubility of this construct wasfurther observed in electrophoresis assays (Figure S5).

Peptide-DNA 4 was synthesized following the same click chemistryprotocol as above for peptide-DNA 3. This product was soluble inaqueous solution and was characterized via agarose gel electrophoresisand atomic force microscopy imaging.

Digestion of Peptide-DNA 3 with Trypsin. A total of 5 pmol ofpeptide-DNA 3 was suspended in 30% DMSO in 10 mM Tris pH 8.5in the presence of 7.5 μg trypsin (Sigma) in 100 μL of total reactionvolume. A total of 5 pmol of unlabeled DNA isolated from the controlreaction was similarly treated as a negative control. As positivecontrols, various concentrations of azido-PEG4-peptide 2 wereproteolyzed by trypsin under the same reaction conditions. Allreactions were performed at 37 °C. Cleavage of the peptide results inrelease of the dinitrophenyl quencher; thus, reaction progress wasfollowed by probing for methoxycoumarin emission at 400 nmfollowing excitation into the red edge of its absorbance profile at 360nm in a fluorescence plate reader (BioTek Synergy). Measurementsshown are an average of two replicates. In the presented data, thediscontinuity at 20 min was a systematic error among all fluorescentreplicates in the plate reader measurement and coincided withoperator intervention to change the time scale of kinetic datarecording.

Atomic Force Microscopy Imaging. Peptide-DNA 4, alkyne-DNA, and DNA were deposited on freshly cleaved mica surfaces basedon a published protocol.39 Briefly, the samples were diluted toapproximately 0.5 nM in 10 mM Tris pH 8.0, 2 mM NaCl, and 2 mMMgCl2. Samples were applied to mica, and after 10 min, NiCl2 at a finalconcentration of 2.5 mM was added. After a further minute, the micawas rinsed briefly with water and dried with a nitrogen flow. AFMimages were obtained in tapping mode on an Asylum Research,

Biomacromolecules Article

dx.doi.org/10.1021/bm501109p | Biomacromolecules 2014, 15, 4065−40724066

Cypher AFM using AC160TS tips with a nominal spring constant of40 N/m.In the images, alkyne-DNA or peptide-DNA constructs were fitted

with parametric splines using custom software.40 Only individualmolecules that did not touch or cross were selected for analysis. Heightinformation was extracted from section analyses, which wereperformed using the AFM software at random locations over a largenumber of molecules. Persistence lengths were found by determiningthe mean squared end-to-end distance ⟨R2⟩ as a function of inner

contour length L, and least-squares fitting to find the persistencelength p according to41

⟨ ⟩ = − − −⎛⎝⎜⎜

⎛⎝⎜⎜

⎛⎝⎜

⎞⎠⎟⎞⎠⎟⎟⎞⎠⎟⎟R pL

pL

Lp

4 12

1 exp2

2

(1)

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

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-1,7-diynyl)-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.



avoid the underestimation of p that can occur when polymers adsorbbefore fully equilibrating on the 2D surface, a fractional dimension of2.5 ± 0.5 was considered.42,43 The assumption of lack of complete 2Dequilibration was further supported by analysis of the kurtosis of theθ(L) distribution (where θ is the angle formed by 2 tangents separatedby L).41

■ RESULTS AND DISCUSSION

Preparation and Characterization of Alkyne-DNA.PCR amplification of pUC19 plasmid was used to generate a968 bp (“1 kbp”) alkyne-labeled DNA, in which ∼1/4 of thenucleotide bases displayed an alkyne moiety. This was achievedby completely replacing dTTP with C8-alkyne-dUTP (Figure1a) during amplification. Both Pwo and KOD XL DNApolymerases36 incorporated C8-alkyne-dUTP into 1 kbp DNA,and using KOD XL polymerase, we were able to generatealkyne-DNA up to ∼3 kbp in length (Supporting Information).As previously observed, relative to control samples alkyne-labeled DNA migrated more slowly in agarose gel electro-phoresis (Figure S1).36

For use as versatile modular building blocks, the ends of thealkyne-DNA products incorporated restriction sites to facilitatesubsequent ligation into diverse DNA platforms. PspOMI andEagI restriction endonucleases were chosen because theirrecognition sequences lack adenine and thus should remainunmodified following amplification. By designing primers foramplification in which the first alkyne-dUTP was incorporated>5 basepairs from the recognition site (Supporting Informationand Figure S2), the resulting products were cleaved by theserestriction enzymes and subsequently ligated into longerproducts.30 These results demonstrate the ability to create,via ligation to orthogonally labeled (or unlabeled) DNA,constructs presenting distinct target sites for interaction andvisualization.Preparation and Characterization of Peptide-DNA.

From this alkyne-DNA, a peptide-DNA hybrid was prepared,displaying an internally quenched fluorogenic peptide, Mca-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2 (1). Trypsinhydrolysis provides an assay for the retention of peptideproperties of this commercially available peptide, demonstrat-ing both that the peptide has been linked to the DNA via thelysyl side chain and that the peptide remains accessible toproteolysis.Covalent attachment of the peptide to alkyne-DNA was

accomplished first by modifying the ε-amino group of thepeptide’s lysyl side chain with NHS-PEG4-azide to introducethe azide moiety necessary for copper-mediated triazoleformation (Figure 1b). The PEG spacer was included toreduce potential steric hindrance to dense peptide incorpo-ration along the DNA and to enhance trypsin accessibility tothe DNA-linked peptides. Azide modification was confirmed bymass 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 reactionbetween 1 kbp alkyne-DNA and unmodified peptide 1 wasperformed as a control, and in both reactions, tris-(benzyltriazolylmethyl)-amine (TBTA) was added to minimizeCu(I)-induced DNA strand breaks.44 After 2 h at roomtemperature, a loose yellow precipitate was observed in thereaction sample. Following ethanol precipitation, the productwas resuspended in either DMSO or 10 mM Tris, pH 8.5. Duelikely to the high local concentration of hydrophobic groups(peptide and dye molecules) arising from their close spacing on

the DNA (see below), the reaction product had only limitedaqueous solubility. Nonetheless, it was amenable to somespectroscopic-based characterization, demonstrating the cova-lent attachment of peptides to DNA.Absorption spectra show that the peptides were linked

successfully to DNA (Figures 2 and S6): absorbance featuresfrom 320 to 420 nm are due to the peptide’s methoxycoumarinand dinitrophenyl groups,45 while the strong peak at 260 nmindicates the presence of DNA.

Peptide incorporation efficiency was assessed in two differentways. First, the concentrations of DNA and peptide wereestimated from the optical densities, and the ratio of theseconcentrations was taken. As observed previously, the presenceof alkyne groups broadens and slightly shifts the characteristicDNA absorbance peak.36 Values for extinction coefficients usedwere as follows: 968 bp DNA, ε260 = 1.28 × 107 M−1 cm−1

(assumed to be unchanged from unmodified DNA); methox-ycoumarin, ε324 = 12900 M−1 cm−1; dinitrophenyl, ε363 = 15900M−1 cm−1, ε410 = 7500 M−1 cm−1.45 This method resulted in anestimate of ∼75 peptides/DNA when applied to the constructresuspended in aqueous conditions (Figure 2a). However, this

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



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-DNAhybrid could contribute scattering to the optical density at 260nm; (iii) Control experiments showed that coprecipitatedTBTA also contributes to absorbance readings at 260 nm; (iv)If the densely incorporated peptide dyes form aggregates, as hasbeen observed for densely labeled cyanine-DNA,38 a blue-shiftin absorbance would occur, contributing further optical densityat 260 nm and concomitantly decreasing absorbance in thefluorophore and quencher monomer absorption peaks. Forcomparison, the same analysis was performed on peptide-DNAresuspended in DMSO (Figure S6). Assuming the sameextinction coefficients, this resulted in an estimate of ∼450peptides/DNA, stoichiometric incorporation of peptide.However, this value is likely to overestimate the labelingdensity, primarily due to the probable underestimate of DNAconcentration in solution due to the strong absorbance byDMSO in the same spectral region.Alternatively, the extent of peptide functionalization of DNA

was estimated by comparing the amount of unreacted azido-peptide 2 remaining in the ethanol supernatant followingprecipitation of peptide-DNA 3 with the control sample(Figure 2b). The difference between the absorbance readings ofthe two samples corresponds to the amount of azide-modifiedpeptide that reacted with the alkyne-DNA. Using measuredabsorbances at 324 nm gave an estimate of 264 peptides/DNA,but this value is unreliable because of contributions fromabsorbance of other soluble species at shorter wavelengths.Measured absorbances attributable to dinitrophenol at twodifferent wavelengths each provided an incorporation density of∼300 peptides/DNA (292 peptides/DNA at 363 nm; 306peptides/DNA at 410 nm). This wavelength range has noobvious contributions from other species, and because theabsorbance of peptides was measured in free solution, it wasnot biased by possible spectral shifts arising from dense packingas may occur with the DNA-tethered peptides.38 For thesereasons and the self-consistency of results between the twowavelengths, this labeling density was taken as the most reliableestimate 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 approximatelyone peptide/nm along the DNA backbone.Control over Peptide Density in Peptide-DNA. Peptide

density can readily be adjusted by modifying the ratio of azido-peptide to alkyne-DNA during click chemistry coupling. Tocircumvent problems of aqueous solubility, these experimentsused a more hydrophilic peptide (acetyl-Ser-Asp-Lys-Pro-OH)lacking a fluorophore/quencher pair, which resulted in solublehybrid constructs. Following coupling of the peptide to theDNA as above, purified peptide-DNA 4 was observed byagarose gel electrophoresis (Figure 3). As the ratio of azido-peptide/alkyne-DNA increased, the resulting peptide-DNAband shifted toward higher molecular weight as expected.The monotonic decrease of mobility and the tightness of theband on the gel demonstrate control over the average peptidedensity and show that the distribution of peptide densitieswithin a given sample is narrow. The ease with which peptidedensity on DNA can be controlled lends itself well tooptimization of labeling density for each desired specificfunction.Enzymatic Addressability of Peptides in Densely

Labeled Peptide-DNA. For incorporation of peptide-DNA

hybrid constructs into functional materials or devices, theretention of peptide and DNA properties is essential forindependent control over structure and function. To establishwhether peptide properties are preserved in densely labeledconstructs, the ability of trypsin to hydrolyze peptide-DNA wasinvestigated.Fluorogenic peptide-DNA 3 was used to assess the

accessibility of tethered peptides to trypsin, through a kineticfluorescence assay. Because peptide hydrolysis results in releaseof the dinitrophenyl quencher from the peptide-DNA construct3, the fluorescence increase seen following addition of trypsin(Figure 4) indicates successful enzymatic cleavage of tetheredpeptides. These assays were conducted in a buffer containing30% DMSO, conditions that maintained trypsin activity (FigureS7), and in which peptide-DNA 3 only very gradually fell out ofsolution with time (Figure S8). In these conditions, fluorogenicpeptide-DNA was hydrolyzed at a slower rate than freepeptides, which at comparable concentrations were cleavedalmost immediately in this assay (Figure 4). This difference incleavage rate may arise from the hindered accessibility of boundpeptides resulting from reduced solubility of this denselylabeled construct. It is likely that a lower density of peptides onthe DNA would result in soluble products, since peptides andDNA are each soluble in aqueous conditions; it is at the highlocal density of peptides in peptide-DNA 3 that insolubilityresults. This would eliminate the need for DMSO, but here, thechoice to work with the densely labeled construct was driven bythe desire to obtain sufficiently strong fluorescence signal toclearly establish peptide cleavage.The extent and rate of peptide cleavage from this construct

was determined by fitting the time-dependent increase influorescence intensity to a single-exponential model:

= − −c t c Ae( ) (1 )k t0

cl (2)

Here, c(t) represents the time-dependent concentration ofcleaved peptides, c0 is its asymptotic limit and kcl is the rate ofcleavage. The constant A is included to account for a finiteamount of fluorescence signal at t = 0. To relate fluorescenceintensity to the amount of peptides cleaved, and to account forthe gradual photobleaching of fluorophores, the time at whichthe peptide-DNA hybrid curve crossed each of the free peptidecurves 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 trypsinof c0 = 4.0 ± 0.4 μM (Figure 4b). As an alternative approach,photobleaching data of free peptides were fit to a global decaycurve, which provided estimates of additional c(t) points for

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



cleavage of the tethered peptide (Supporting Information andFigure S9). This analysis did not change the asymptoticcleavage (c0 = 4.0 ± 0.2 μM) or rate constant (kcl = 0.023 ±0.004 min−1). Thus, for a 50 nM hybrid, these estimatescorresponded to ∼80 cleaved peptides/peptide-DNA hybrid(∼27% reaction efficiency). This likely underestimates thereaction efficiency for two reasons: (i) the concentration ofhybrid is based on the amount of DNA reacted with peptide,which overestimates its final recovered concentration due tosmall losses during ethanol precipitation and recovery and dueto reduced solubility in the cleavage reaction; and, moreimportantly, (ii) additional quenching of Mca fluorescence mayarise from nearby peptide moieties along the DNA backbone,such that 4.0 μM (from comparsion with cleaved peptide insolution) underestimates the concentration of cleaved peptidesin the hybrid. Nonetheless, this assay demonstrated that asignificant portion of tethered peptides remains accessible toprotease recognition and cleavage. They can thus be consideredas addressable entities when incorporated along the DNAbackbone, where they could serve for example as substrates fordesigned proteolytic molecular motors.7,46

Retention of DNA Mobility and Persistence Length inDensely Labeled Peptide-DNA. A further requirement formodular design is the retention of DNA properties in thesehybrid constructs. Electrophoretic migration assays (Figure 3)showed that DNA migrates as expected in an agarose gel, withits mobility reduced in a titratable manner with the addition ofpeptide groups. To examine the effect of covalent modificationson the flexibility of DNA as a polymer, relevant fornanomanipulation and for function as a modular scaffold forhydrogels, 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 nosignificant difference between alkyne- and peptide-DNA 4,suggesting that these short tethered peptides do not addenough 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 unmodifiedDNA persistence length in standard buffer solutions41,47 andwith previous reports on shorter fluorophore-labeled DNA.37

This short peptide is expected to contribute a net negativecharge to the hybrid construct, and thus might be expected toincrease the persistence length of the DNA through increasedelectrostatic repulsion. However, the Debye length of 2 nm inthe 20 mM ionic strength of the buffer solution used in theseexperiments is comparable to the length of the PEG4 spacerarm used to link the peptide to the DNA, and thus, it is likelythat the unchanged persistence length results from sufficientelectrostatic screening by this buffer. Thus, our results showthat dense functionalization with alkynes and peptides does notdisrupt DNA’s mechanical properties and should permit facilehandling, characterization, and assembly with establishedtechniques.

■ CONCLUSIONSIn this work, we constructed densely labeled 1 kbp peptide-DNA hybrid constructs and demonstrated that they retainpeptide and DNA properties. The use of a commerciallyavailable DNA polymerase36 to generate peptide-presenting,long DNA substrates suggests the compatibility of thisfunctionalization approach with current methods of DNA-based hydrogel formation.22,24 Moreover, the ease with whichthese densely functionalized hybrid structures were constructedcontrasts with the more stringent demands of alternativeapproaches for DNA labeling such as origami, where numerousstrands must each be labeled in a specific location to present ahigh local concentration of targets, and where the labelingdensity is limited to approximately one modification per 6 nm.9

The density of ∼1 peptide/nm achieved here is significantlyhigher. Because the locations of modifications here arestochastic rather than prescribed as in origami, this representsan average peptide density along the DNA backbone and maybe even greater in alkyne-rich regions.There are many potential applications of these extended

linear DNA hybrids of tunable peptide density. The use ofmodular peptide-DNA building blocks, in which the structureand function of each is independently maintained uponincorporation into hybrid structures, suggests the possibilityof using both DNA and peptide as orthogonally tunablescaffolds in a given material, enabling the independent drivingand tuning of structure and function by both components. Theability to control average peptide density, as shown for peptide-

Figure 4. (a) Incubation of peptide-DNA 3 with trypsin resulted ingradually increasing fluorescence from methoxycoumarin (solid redtrace), resulting from cleavage of the tethered peptide and release ofthe dinitrophenyl quencher into solution. Incubation of control DNAwith 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 forphotobleaching in solution, from the intersection times of the freepeptide cleavage curves with the peptide-DNA curve (arrows), theconcentration of peptide cleaved at each time from the hybrid wasestimated. (b) Concentration of peptides cleaved from peptide-DNA 3as 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 μMcleaved peptides.



DNA 4 in this work, may prove particularly useful when tuningproperties such as hydrogel cross-link density and backbonesolubility at high concentrations. To enhance their versatility,these constructs could be further incorporated into morecomplex structures through ligation to orthogonally labeledDNA, presenting diblock or multiblock polymeric systems.30,48

The use of DNA as a one-dimensional backbone forassembly has applications in synthetic molecular motordesign,4−7,30,31,46 where its presented cleavable peptides couldbe used to direct biased motion.7,31,46 The advantages of thisapproach over alternative linear templating strategies31 includethe growing variety of techniques available for the nano-manipulation of DNA,32−35 which should be directly applicableto 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 anddrug delivery, where presentation of a high local concentrationof targets can strongly enhance signal through polyvalency andcooperativity.3,28,29,49 In summary, the ability to control in afacile manner the density of peptide modifications on DNA hasthe potential to open the door to a wide variety of applicationsin nanoscale device design, biosensing, and responsive materialsdevelopment.

■ ASSOCIATED CONTENT

*S Supporting InformationSupporting text, Tables S1 and S2, and Figures S1−S9. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by a research grant from the HumanFrontier Science Program (Grant No. RGP31/2007), byNSERC Discovery Grants (Forde, Li), and the SwedishResearch Council (Linke). We thank Andrew Wieczorek foruseful discussions and Dr. Hongwen Chen for conducting theESI-MS in the laboratory of Dr. Robert Young.

■ REFERENCES(1) Feldkamp, U.; Sacca, B.; Niemeyer, C. M. Angew. Chem., Int. Ed.2009, 48, 5996.(2) Woolfson, D. N.; Mahmoud, Z. N. Chem. Soc. Rev. 2010, 39,3464.(3) Roh, Y. H.; Ruiz, R. C. H.; Peng, S. M.; Lee, J. B.; Luo, D. Chem.Soc. Rev. 2011, 40, 5730.(4) Bath, J.; Green, S. J.; Turberfield, A. J. Angew. Chem., Int. Ed.2005, 44, 4358.(5) Green, S. J.; Bath, J.; Turberfield, A. J. Phys. Rev. Lett. 2008, 101,238101.(6) Bromley, E. H. C.; Kuwada, N. J.; Zuckermann, M. J.; Donadini,R.; Samii, L.; Blab, G. A.; Gemmen, G. J.; Lopez, B. J.; Curmi, P. M.G.; Forde, N. R.; Woolfson, D. N.; Linke, H. HFSP J. 2009, 3, 204.(7) Lund, K.; Manzo, A. J.; Dabby, N.; Michelotti, N.; Johnson-Buck,A.; Nangreave, J.; Taylor, S.; Pei, R.; Stojanovic, M. N.; Walter, N. G.;Winfree, E.; Yan, H. Nature 2010, 465, 206.(8) Simmel, F. C. Curr. Opin. Biotechnol. 2012, 23, 516.

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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(9) Fu, J. L.; Liu, M. H.; Liu, Y.; Yan, H. Acc. Chem. Res. 2012, 45,1215.(10) Sacca, B.; Niemeyer, C. M. Angew. Chem., Int. Ed. 2012, 51, 58.(11) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D.A. Science 1998, 281, 389.(12) Cao, Y.; Li, H. Chem. Commun. (Cambridge, U.K.) 2008, 4144.(13) Banta, S.; Wheeldon, I. R.; Blenner, M. Annu. Rev. Biomed. Eng.2010, 12, 167.(14) Lv, S.; Dudek, D. M.; Cao, Y.; Balamurali, M. M.; Gosline, J.; Li,H. Nature 2010, 465, 69.(15) Hudalla, G. A.; Sun, T.; Gasiorowski, J. Z.; Han, H.; Tian, Y. F.;Chong, A. S.; Collier, J. H. Nat. Mater. 2014, 13, 829.(16) Sun, F.; Zhang, W.-B.; Mahdavi, A.; Arnold, F. H.; Tirrell, D. A.Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 11269.(17) Li, X.; Kuang, Y.; Lin, H.-C.; Gao, Y.; Shi, J.; Xu, B. Angew.Chem., Int. Ed. 2011, 50, 9365.(18) Chen, P.; Li, C.; Liu, D. S.; Li, Z. B. Macromolecules 2012, 45,9579.(19) Dooley, K.; Kim, Y. H.; Lu, H. D.; Tu, R.; Banta, S.Biomacromolecules 2012, 13, 1758.(20) Park, N.; Um, S. H.; Funabashi, H.; Xu, J.; Luo, D. Nat. Mater.2009, 8, 432.(21) Aldaye, F. A.; Senapedis, W. T.; Silver, P. A.; Way, J. C. J. Am.Chem. Soc. 2010, 132, 14727.(22) Lee, J.; Peng, S. M.; Yang, D. Y.; Roh, Y. H.; Funabashi, H.;Park, N.; Rice, E. J.; Chen, L. W.; Long, R.; Wu, M. M.; Luo, D. Nat.Nanotechnol. 2012, 7, 816.(23) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487.(24) Qi, H.; Ghodousi, M.; Du, Y.; Grun, C.; Bae, H.; Yin, P.;Khademhosseini, A. Nat. Commun. 2013, 4, 2275.(25) Discher, D. E.; Janmey, P.; Wang, Y.-l. Science 2005, 310, 1139.(26) Janmey, P. A.; Weitz, D. A. Trends Biochem. Sci. 2004, 29, 364.(27) Murphy, W. L.; McDevitt, T. C.; Engler, A. J. Nat. Mater. 2014,13, 547.(28) Martinez-Veracoechea, F. J.; Leunissen, M. E. Soft Matter 2013,9, 3213.(29) Zhao, W.; Cui, C. H.; Bose, S.; Guo, D.; Shen, C.; Wong, W. P.;Halvorsen, K.; Farokhzad, O. C.; Teo, G. S. L.; Phillips, J. A.; Dorfman,D. M.; Karnik, R.; Karp, J. M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109,19626.(30) Kovacic, S.; Samii, L.; Woolfson, D. N.; Curmi, P. M. G.; Linke,H.; Forde, N. R.; Blab, G. A. J. Nanomater. 2012, 2012, 109238.(31) Cha, T.-G.; Pan, J.; Chen, H.; Salgado, J.; Li, X.; Mao, C.; Choi,J. H. Nat. Nanotechnol. 2014, 9, 39.(32) Farre,́ A.; van der Horst, A.; Blab, G. A.; Downing, B. P. B.;Forde, N. R. J. Biophotonics 2010, 3, 224.(33) Fazio, T.; Visnapuu, M.-L.; Wind, S.; Greene, E. C. Langmuir2008, 24, 10524.(34) Gorman, J.; Fazio, T.; Wang, F.; Wind, S.; Greene, E. C.Langmuir 2009, 26, 1372.(35) Reisner, W.; Beech, J. P.; Larsen, N. B.; Flyvbjerg, H.;Kristensen, A.; Tegenfeldt, J. O. Phys. Rev. Lett. 2007, 99, 058302.(36) Gierlich, J.; Gutsmiedl, K.; Gramlich, P. M. E.; Schmidt, A.;Burley, G. A.; Carell, T. Chem.Eur. J. 2007, 13, 9486.(37) Ramsay, N.; Jemth, A.-S.; Brown, A.; Crampton, N.; Dear, P.;Holliger, P. J. Am. Chem. Soc. 2010, 132, 5096.(38) Smith, D. A.; Holliger, P.; Flors, C. J. Phys. Chem. B 2012, 116,10290.(39) Lysetska, M.; Knoll, A.; Boehringer, D.; Hey, T.; Krauss, G.;Krausch, G. Nucleic Acids Res. 2002, 30, 2686.(40) Lamour, G.; Kirkegaard, J.; Li, H.; Knowles, T.; Gsponer, J.Source Code Biol. Med. 2014, 9, 16.(41) Rivetti, C.; Guthold, M.; Bustamante, C. J. Mol. Biol. 1996, 264,919.(42) Smith, J. F.; Knowles, T. P. J.; Dobson, C. M.; MacPhee, C. E.;Welland, M. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15806.(43) Lamour, G.; Yip, C. K.; Li, H.; Gsponer, J. ACS Nano 2014, 8,3851.

(44) Gierlich, J.; Burley, G. A.; Gramlich, P. M. E.; Hammond, D. M.;Carell, T. Org. Lett. 2006, 8, 3639.(45) Knight, C. G.; Willenbrock, F.; Murphy, G. FEBS Lett. 1992,296, 263.(46) Samii, L.; Blab, G. A.; Bromley, E. H. C.; Linke, H.; Curmi, P.M. G.; Zuckermann, M. J.; Forde, N. R. Phys. Rev. E 2011, 84, 031111.(47) Bustamante, C.; Marko, J. F.; Siggia, E. D.; Smith, S. Science1994, 265, 1599.(48) Hili, R.; Niu, J.; Liu, D. R. J. Am. Chem. Soc. 2013, 135, 98.(49) Stephanopoulos, N.; Ortony, J. H.; Stupp, S. I. Acta Mater.2013, 61, 912.



construction and characterization of kilobasepair densely

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