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pubs.acs.org/bc

Self-Assembly Properties and Dynamics of Synthetic ProteoNucleic
Building Blocks in Solution and on Surfaces
Aude Laisne,† Maxime Ewald,‡ Toshio Ando,§ Eric Lesniewska,‡ and Denis Pompon*,††Centre de Genetique Moleculaire, CNRS, UPR3404, Avenue de la Terrasse, F91190 Gif-sur-Yvette, FranceInstitut Carnot Bourgogne, UMR CNRS 5209, University of Bourgogne, F21078 Dijon, France§
Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan‡bS Supporting Information
ABSTRACT: Synthetic proteonucleic structures (PDNAs)
encompassing a single-stranded DNA sequence covalently attached to a redox protein domain able to interact with surface ormatrix were designed and characterized. They constitute versatilebuilding blocks alternative to regular DNA for creating scaﬀolds
with optical, electrical, or catalytic properties. PDNAs self-assemble in the presence of complementary oligonucleotides, to form a
network of protein domains linked by double-stranded DNA
segments. Electrophoretic and hydrodynamic behaviors of
PDNAs and corresponding DNA were compared under electrophoresis and gel ﬁltration conditions. Hybridization rates between small and large assemblies were characterized by rapidmixing experiments. Results showed that the protein part signiﬁcantly contributes to hydrodynamic behaviors of structures but marginally aﬀects the conformation and hybridization propertiesof the nucleic domain. PDNA metal-mediated complexes with nitriloacetate-modiﬁed phospholipids can diﬀuse and interact at thesurface of vesicles or supported membranes. Surface plasmon resonance analysis of membranePDNA interactions indicated thattwo protein units are required to allow stable surface association and that surface occupancy constrains assembly sizes. High-speed
atomic force microscopy illustrated rapid lateral diﬀusion of assemblies on mica, revealing transient association between
noncomplementary PDNA extremities and frequent trapping by surface defects. Regularly organized protein domains werevisualized using a larger DNA framework.

’ INTRODUCTION
The use of a synthetic DNA scaﬀold to build mono- or
multidimensional nano-objects with fully predictable shapes
rapidly developed as an important ﬁeld of synthetic biology.17
However, the limited range of functionalities that can be assembled using only nucleic acid tiles raised the question of theposition speciﬁc incorporation of protein block in such scaﬀolds.8
One way involved the DNA binding domain or the recognition of
modiﬁed bases (for example biotin) by speciﬁc protein domains(avidin, antibodies, etc.).811 Reciprocally, alternate approaches
proﬁt from the recognition of speciﬁc protein domains byaptamer structures embedded into the DNA scaﬀold.12 However,these two methods suﬀer strong limitations because they require
fusion of a large protein recognition domain to functional parts ofinterest or restrict the approach to structures for which suitableaptamers can be created. ProteinDNA recognition based on
chemically mediated approaches (such as NTAmetal complexes) can also be considered, but either gives rise to reversible
association or lacks speciﬁcity.13,14
PDNAs (proteinDNA structure), which are artiﬁcial structuresthat consist of a protein domain covalently linked to a singler 2011 American Chemical Society

stranded DNA (ssDNA) part through synthetic structures, constitute an attractive alternative.15,16 The protein part of PDNA can bechosen to carry various types of functions such as surface interaction,binding, enzymatic, optical, or electrical properties, potentiallycreating nanostructures that can be used as biosensors17,18 or active
molecular devices. PDNA tiles can self-assemble in solution or be
attached to a matrix in the presence of complementary ssDNA.19
However, little is known about the stability and physicochemical andstructural features of such objects or the ways they interact together
compared to regular DNA tiles. This is particularly critical for
deﬁning methods adapted for speciﬁcally incorporating thesebuilding blocks into DNA scaﬀolds like origami6 or other DNAbased supramolecular assemblies.20
This paper extends synthesis approaches for such PDNA
building blocks and analyzes their physicochemical and assembly
properties in comparison to their corresponding pure nucleotidic
parts, at both global and single-molecule levels.
Received: May 2, 2011
Revised:July 8, 2011
Published: August 19, 2011
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Bioconjugate Chemistry’ EXPERIMENTAL PROCEDURES
Synthesis of the PDNA Building Block. Oligonucleotides(Eurogentec) presented in section 1 of the Supporting Information were purified by ion-pairing reverse phase chromatography
as previously described.21 PDNAs were prepared by hemisynthesis from engineered human cytochrome b5 (b5) as described in
sections 2 and 3 of the Supporting Information and previous
work.16,17,19 Briefly, a 30 - or internally amino linker-modified
oligonucleotide was covalently attached through a disulfide bondto a modified b5 containing a unique Cys residue and a His4
C-terminal tag. During the synthesis, the amino linker of the
ssDNA was coupled to a second linker terminated by a thiopyridine-protected thiol using NHS chemistry. A disulfide bond was
finally formed by reacting the activated linker end with theunique Cys residue of the protein. The resulting products were
purified to homogeneity by a combination of ion exchange andaffinity chromatography, taking advantage of the protein His tag.
PDNA Thermal Stability. Experiments were performed in
20 mM Na/K phosphate buffer (pH 7.4) using PDNA or b5
concentrations in the 10 μM range. Thermal denaturation wasmonitored on a Lambda 2 (Perkin-Elmer) spectrophotometerusing increasing temperature steps of 4 °C. Spectra were recorded between 260 and 600 nm immediately after each temperature stabilization at a rate of 120 nm/min. Denaturation
curves for native, engineered b5, and PDNA were calculated fromthe changes in the 413 nm absorbance.
Phospholipid Vesicle Preparation. 1,2-Dimyristoyl-rac-glycero-3-phosphocholine (DMPC) was purchased from SigmaAldrich and 1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} (nickel salted) (DOGSNTA) from Avanti Polar Lipids. A chloroform solution containing 1 mg of a DMPC/nickel DOGS-NTA mixture (90:10 molar
ratio) was evaporated into a glass vial under a nitrogen flow, andthe residue was dried under vacuum for 2 h. Phospholipids were
hydrated overnight at 4 °C with 1.4 mL of 1 PBS buffer (SigmaAldrich), and the mixture was sonicated until a clear solution
containing a total phospholipid concentration of 1 mM was
obtained. The vesicles were extruded 19 times through polycarbonate membranes with 100 nm pores (Liposofast, Avestin).
Synthesis of Defined PDNA Assemblies. A detailed description is given in section 8 of the Supporting Information. Briefly,
hybridizations of DNA and PDNA tiles were performed at 24 °C
in 1 PBS buffer using a slight molar excess of DNA compared to
PDNA. The constructs were purified from excess DNA either by
association of IMAC and Sephadex G25/G50 chromatography
or by HPLC size exclusion chromatography. The first method
involved binding to an IMAC column (0.5 cm  1.5 cm) andelution with a buffer supplemented with 30 mM histidine. EDTA
(final concentration of 10 mM) was added to the eluate prior
it being loaded onto a Sephadex G25 gel filtration column(0.5 cm  1.5 cm) equilibrated with 1 PBS buffer. Alternatively, purification was performed by size exclusion chromatography (BioSuite 250, 4 μm UHR SEC, 4.6 mm  300 mm,
Waters) at a rate of 0.3 mL/min with 0.25 M NaCl and 0.15 M
NaKPO4 buffer (pH 6.8). DNA Ladders N3233 (Biolabs) wereused for calibration.
Analysis of DNA and PDNA Hybridization Kinetics by
Rapid-Mixing Experiments. Hybridization of simple DNA
and PDNA tiles and preformed dimers was analyzed in rapidmixing experiments by monitoring the hypochromic effect associated with the formation of double-stranded structures. Kinetic

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constants were calculated by fitting of the traces as described
previously.21
Gel Filtration and Electrophoresis Methods. HPLC gel
filtration analysis was performed on a Waters BioSuite 250
column (4 μm UHR SEC, 4.6 mm  300 mm) at a rate of
0.3 mL/min with 0.25 M NaCl and 0.15 M NaKPO4 buffer (pH6.8). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) analysis was performed using 4 to 12%
NuPAGE Novex Bis-Tris polyacrylamide gels run at 24 °C and120 V. Protein parts of PDNA were revealed by fast Coomassie
blue staining and calibrated using the Mark12 Unstained standard from Invitrogen. Agarose gel analysis was performed using
3.5% NuSieve GTG Agarose from Tebu-bio in 1 TBE run at
50 V and 4 °C. DNA Ladders N3233 (Biolabs) were used forcalibration. Agarose gel electrophoresis patterns were deconvoluted using an optimized expansion factor of 1.5 and band
quantified as described previously for the analysis of regularDNA polymers.21
Surface Plasmon Resonance (SPR) Methods. SPR experiments were performed on a SPRi-Lab+ array system (Genoptics)
using 1 PBS as running buffer at a flow rate of 230 μL/min
and 24 °C. Gold layer-covered prisms were cleaned by gentle
sonication in a hot acid Pyranha solution (equal volume of 30%hydrogen peroxide and pure sulfuric acid), abundantly rinsed
with Milli-Q water, and stored in pure HPLC grade acetonitrile
until they were used. Experiments were performed following
phospholipid vesicle fusion either on bare gold (lipid bilayer) or
on a self-assembly monolayer of alkyl chains (lipid hemilayer).
For that, the Pyranha-cleaned prism was immersed in a freshly
prepared 1 mM solution of 1-octadecanethiol in ethanol (Sigma
Aldrich) for 18 h and then rinsed with ethanol and water. Freshly
extruded phospholipid vesicles (1 mM into 1 PBS buffer) werefused onto the treated substrate by circulation in the SPR cell at
a flow rate of 50 μL/min for 30 min at 24 °C. An excess
of adsorbed vesicles was removed by washing for 10 min withpure water (hypo-osmotic choc) followed by equilibration with
1 PBS buffer.
High-Speed Atomic Force Microscopy (HS-AFM). A laboratory-built high-speed atomic force microscope described previously was used to visualize the structure.2224 All observations were performed in oscillating contact mode in liquid.
Small cantilevers designed for HS-AFM with a spring constantof 0.10.2 N/m, a resonant frequency of 0.81.2 MHz, and aquality factor of ∼2 in buffer solution were used.25 The cantilever tips were grown by electron beam deposition (SEM Jeol
6500) and further sharpened by oxygen plasma etching (Plasma
cleaner Harrick). The cantilever’s free oscillation peak-to-peakamplitude (A0) was set to 45 nm, while the feedback set point
during imaging was set to ∼0.9 A0. The samples were diluted(210-fold) in an observation buffer (OB) containing 10 mM
MgCl2. A drop (∼2 μL) was deposited on freshly cleaved green
muscovite mica, and the mica was incubated at room temperature for 5 min. Subsequently, the mica surface was immersed ina liquid chamber filled with 60100 μL of OB and subjected to
HS-AFM. Diluted samples were used within a day (storage at
4 °C).

’ RESULTS
Three types of PDNAs including an engineered cytochrome
b5 redox domain and diﬀering by the sequence of their nucleotidic parts and by the grafting position on the sequence of the1825

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Bioconjugate Chemistry

Figure 1. Structures of the PDNA building block, including a b5 core.
The structure is composed of a 39-base ssDNA part (A) attached either
by its 30 -end (as illustrated) or by an internal amino C6 thymidine (notshown) to a synthetic linker (B) coupled to cytochrome b5 (C) by thethiolate extremity of a cysteine at position 24. The His4 C-terminal end(D) of the protein interacts with the nickel ion of an 18:1 DOGS-NTAmodiﬁed lipid (E). The structure at the bottom left shows PDNA blocks
inserted into a dsDNA structure. The structure is ﬂoating on a supported
membrane composed of a phospholipid monolayer covering a selfassembled octadecanethiol monolayer on gold.

Figure 2. Thermal stabilities of b5 folding in wild-type, Cys-modiﬁed,
and corresponding PDNA structures. Protein denaturation was monitored by observation of the spectral changes of the bound heme cofactor
at 413 nm. Denaturation curves were compared for native (2), Cysmodiﬁed b5 (0), and PDNA (b) forms. Typical spectral changes
following incubation of a PDNA at temperatures increasing from 44
to 68 °C in steps of 4 °C are illustrated in inset curves 1 (44 °C) to 7
(68 °C).

protein domain (Figure 1, right) were prepared by hemisynthesis
and puriﬁed to homogeneity (sections 2 and 3 of the Supporting
Information). In these structures, the protein C-terminal His tag

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Figure 3. Comparative rate constants for DNA and PDNA hybridizations. (A) Hybridizing oligonucleotides are indicated inside bars
(matching segments underlined). Dark and light gray boxes refer tohybridization of DNA and corresponding PDNA to half-cDNA, respectively. Second-order rate constants were deduced from measurements of
the hypochromic eﬀect in rapid-mixing experiments. (B) Ratios among
hybridization rates for normal, base-modiﬁed, and protein-coupledoligonucleotides. These ratios are deﬁned by a scheme in which thecircle stands for the protein domain and the NH2 label for the internal basemodiﬁcation. White and black bars correspond to hybridizations involving
the distal and proximal segments of oligonucleotides, respectively.

plays an important role in permitting the formation of a metalchelate linkage between the PDNAs and Ni-NTA groups
attached to either a polymer matrix, modiﬁed phospholipids, orfunctionalized surfaces. Lateral diﬀusion of anchored PDNAs
remains possible either by stepwise release and rebinding of themetal chelate or, in the case of vesicles or a supported membrane,
through lipid diﬀusion. Hybridization of the PDNA singlestranded part to complementary nucleic acid generates a morerigid double-stranded DNA (dsDNA) structure (Figure 1,
left) that, depending on sequences, could self-organize into awide range of nucleoproteic networks. Such structures canalternatively be made by substituting the cytochrome b5 domain
with any other protein of interest provided that a single grafting
point can be engineered at the surface.
Stability and Hybridization Properties of the PDNA Building Block in Solution. The termal stability of the PDNA protein
domain under conditions required for hybridization reactions is
critical for the maintenance of the functional integrity andparticularly redox properties. This was analyzed in solution bymonitoring the temperature-dependent disruption of the cytochrome b5 absorption spectra, which tightly depend on the ironcoordination by two histidine residues of the protein. Results
indicated that the reversible denaturation of the protein part of
the PDNA is observed only above 55 ( 2 °C, a value identical tothat found for the DNA-free engineered protein and slightly
lower (23 °C) than that for the native protein (Figure 2).
Oxidoreduction properties of the cytochrome b5 domain in
PDNA structures were found to be conserved. An identicaltransition in the absorption spectra, which is characteristic of
the redox change of the histidine-coordinated iron porphyrin,
was observed upon electron transfer in the native protein and on
PDNA structures. Thus, the presence of the ssDNA part has no
significant effect on the stability and redox properties of theprotein domain. The thermal stability of the protein is sufficiently
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Bioconjugate Chemistry

Figure 4. Size exclusion chromatography characterization of short
PDNA assemblies. Structures depicted were analyzed on a HPLC gel
ﬁltration column (detection at 413 and 254 nm) and apparent molecularmasses calculated using dsDNA as molecular mass standards. Apparent
molecular masses (expressed in base pairs) were predicted using theformula molecular mass (base pairs) = 10P + 0.5S + D, where P, S, and D
stand for the number of protein unit(s) (b) and ss- and dsDNA bases(arrows), respectively. The inset shows experimental peak shapes forillustrated constructs.

high to allow hybridization to proceed under classical conditions(55 °C) for oligonucleotides.
Reciprocally, the eﬀect of the protein domain on the hybridization properties of the DNA domain was investigated forstructures in which covalent grafting was performed either
internally or at the 30 -end of the oligonucleotidic part. Monitoring of hypochromic eﬀects was used to analyze the relative rates
of hybridization of the diﬀerent PDNAs with complementary
ssDNA sequences. To discriminate between hydrodynamic
and protein-related microenvironment eﬀects, hybridization
properties were analyzed on both the proximal and the distalsides (30 - and 50 -halves, respectively) of the nucleic part of PDNAs.
In addition, the potential inﬂuence of the nature (ds- vs ssDNA)of the region ﬂanking the hybridization site was investigated.
During hybridization of PDNAs, protein absorption at 260 nm
represents less than 10% of the nucleic acid absorbance and does
not contribute to spectral changes. Second-order hybridization rate
constants ranging from 1.9 to 7 μM1 min1 were observed(Figure 3), while the lengths of the hybridizing segments were
almost identical (1920 bases) in all experiments. Comparison of
hybridization rates for DNA and 30 -end-grafted PDNA with an
identical sequence indicated that protein-dependent modulation ofrates is limited with regard to the eﬀects related to the sequence ofhybridizing segments (Figure 3A). Moreover, analysis of therelative rates of hybridization events occurring in the distal andproximal parts of PDNA conﬁrmed a hardly signiﬁcant inﬂuence ofthe protein domain when grafted at the 30 -end of the nucleic part(labels 1/3 and 2/4 in Figure 3B). In contrast, when the grafting
was performed internally in the nucleic part, a more signiﬁcanteﬀect on rates was observed, which resulted both from thepresence of a modiﬁed base in the hybridizing segment (labels
5/3 and 6/4 in Figure 3B) and the attachment of the protein
domain (labels 7/3 and 8/4). In both cases, distal grafting has alimited inﬂuence compared to proximal grafting, suggesting thatthe eﬀect relates more directly to proteinDNA interaction rather
than to changes in the global hydrodynamic properties in solution.

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Figure 5. SDSPAGE analysis of short PDNA assemblies. (A) Experimental gel, in which the numbering of lanes corresponds to the structure
depicted in the bottom panel. (B) Relation between the observed
molecular mass (calculated on the basis of a protein standard) andthose predicted using the formula molecular mass (kilodaltons) = 15P +0.282S + 0.359D, where P, S, and D stand for the number of protein
unit(s) (b) and ss- and dsDNA bases (arrows), respectively. Structures24 are derived from AA0 -b5 PDNAs, and structure 5 combines AA0 -b5
and BB0 -b5 PDNAs.

Electrophoretic and Chromatographic Properties of
PDNA Assemblies of Increasing Sizes. To further investigate

the relations between hydrodynamic behaviors and structures,
different types of PDNA assemblies were built. The relative
contributions of protein and nucleic domains were analyzed
using a series of constructs built from hybridization-driven
assembly of two PDNA tiles (or corresponding DNA controls),
namely, AA0 -b5 and BB0 -b5 (section 1 of the Supporting
Information). The AA0 PDNA building block was designed tohybridize with the 50 -half of ab0 and the 30 -half of ba0 cDNA,
matching their a and a0 parts, respectively. Similarly, the BB0
PDNA block can hybridize to ba0 and ab0 DNA tiles by matching
their b and b0 parts, respectively. The complementary tiles wereassembled by single-step or sequential (purification of intermediates) hybridizations at low (2545 °C) temperatures
followed by final purification to homogeneity by combinationsof Ni-NTA affinity and gel filtration chromatography to resolve
PDNA assemblies from excess DNA. Corresponding structuresand HPLC gel filtration profiles are illustrated in Figure 4.
Individual constructs migrate during gel filtration as Gaussianshaped bands, allowing easy determination of their relative
mobility under the high-salt buffer conditions required to prevent interaction with the silica matrix of columns. Interestingly,
DNA and protein structural elements contributed in a perfect
additive way to the apparent size calculated on the basis of
dsDNA standards. The individual contributions of the ssDNA(0.5 bp/base), dsDNA (1 bp/bp), and protein (equivalent to 10
bp/unit) parts appeared to be independent of the assemblies
considered. Consequently, the plot of the apparent sizes versus
calculated values for eight different constructs is linear. The 10
bp/unit equivalent contribution of the protein part corresponds
to an apparent molecular mass of 6.6 kDa, a value significantly1827

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Bioconjugate Chemistry

Figure 6. Agarose gel analysis of the apparent molecular mass of PDNA
and DNA assemblies. Structures depicted on the ﬁgure were resolved by
3.5% agarose gel electrophoresis in 1 TBE. Calibration was performed
with dsDNA standards. Molecular masses were predicted using theformula molecular mass (base pairs) = 25P + 0.6S + D for DNADNA
constructs and the formula molecular mass (base pairs) = 25P + 1.55S +D for PDNADNA constructs, where P, S, and D stand for the numberof protein units, ssDNA bases, and dsDNA base pairs, respectively.

smaller than its actual size of 13 kDa, which would correspond tosmaller hydrodynamic radii for proteins compared to those of
dsDNA standards with identical masses. However, this is notconsistent with predicted hydrodynamic radii26,27 that are similar(1.92 nm) for a dsDNA of 13 kDa (20 bp) and for the b5 core
(13 kDa). The difference can be related to the flux-dependenthydrodynamic stretching that is expected to occur and affectdifferentially globular and linear structures in the tightly packed
HPLC gel filtration column.
To gain additional insight, the electrophoretic mobility of thediﬀerent constructs was evaluated under SDSPAGE conditions(Figure 5). Analysis illustrated that coupling of the DNA parts tothe protein induces a strong shift in mobility. The apparent
molecular mass increases from ∼15 kDa for the b5 core alone to
27 kDa for the corresponding single-stranded PDNA, thediﬀerence of 12 kDa being consistent with the molecular mass(∼13 kDa) of the DNA part. This result contrasts with gel
ﬁltration data and could appear surprising as the protein andDNA parts exhibit very diﬀerent permanent charges. However,protein in SDSPAGE is expected to be denatured into an
extended and rather rigid form and negatively charged by theinteracting SDS.28 SDS-denatured protein and dsDNA have thus
some common conformational features that de facto result in
identical electrophoretic mobilities for similar masses. In contrast, the contribution to the apparent molecular mass of the
ssDNA part was consistently found to be ∼50% larger than thatof the dsDNA part for equivalent masses with a lower persistence
length of ssDNA compared to that of dsDNA.
The conformation of PDNA-based structures in solution was
analyzed using nondenaturing agarose gel electrophoresis(Figure 6). An additive contribution to the apparent size
(expressed as base pair equivalents) of PDNA assembly components was observed as described previously with ssDNA, dsDNA,
and protein parts contributing to ∼0.6, 1, and 25 bp equivalents,
respectively (section 4 of the Supporting Information). The
16.5 kDa protein contribution is close to the 15 kDa value
determined by SDSPAGE, while calibration was performed
using dsDNA standards. This feature is the reciprocal of the

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Figure 7. Rapid-mixing analysis of the formation of tile concatemers.
(A) Experimental (gray line) and simulated (black line) kinetic
courses for the copolymerization of the depicted preformed PDNADNA hybrids. (B) Same data for the hybridization of corresponding
DNADNA hybrids. Simulations were performed using rate constantsfrom Figure 3A and parameters from section 6 of the Supporting
Information.

situation found for DNA calibrated on a protein standard using
SDSPAGE. It suggests that the folded b5 core has electrophoretic mobility similar to that of dsDNA with the same mass inagarose gels. Likewise, the relative apparent masses of ss- anddsDNA are similar in the two electrophoretic systems. However,analysis of the electrophoretic mobilities of DNA and PDNA
polymers evidenced that the contribution of ssDNA to theapparent molecular mass diﬀers in PDNA (1.551.78 bp/base)and DNA (0.600.69 bp/base) assemblies (section 4 of the
Supporting Information). The eﬀect is identical with one orseveral protein units per construct and suggests that the presenceof protein aﬀects the contribution to mobility of linked ssDNA
but not of dsDNA (section 4 of the Supporting Information).
This phenomenon was not observed during gel ﬁltration where
the protein is folded but an electrical ﬁeld is absent or during
SDSPAGE where the protein domain is unfolded.
Hybridization Properties of Large PDNA-Based Structures. While hybridization properties of defined small PDNA
and corresponding DNA structures appeared to be very similar,
we questioned the behavior of larger assemblies in which protein
content could significantly impact geometry and steric hindrance
causing topological restriction to base pairing. The model used
was the formation of block copolymers involving regular repeatsof two types of PDNAs. When AA0 and BB0 PDNA were mixed
with half-complementary tiles (ab0 and ba0 ), a polymer featuring
AA0 -BB0 PDNA repeats can be formed. The reaction was firstmonitored using the hypochromic effect resulting from thehybridization of single-stranded sticky ends. Panels A and B of
Figure 7 compare the reaction courses for PDNADNA andDNADNA polymerization, respectively. Individual rate constants described in Figure 3 were used to simulate the reaction
courses using the same modeling approaches21 for PDNA andDNA assemblies. This model assumes that hybridization rates oflarge polymer fragments decrease with a power of 0.57 of
species length for DNA.21,29 For PDNA, an equivalent length was
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Figure 8. Size distribution of DNA and PDNA copolymers.
(A) Agarose gel electrophoresis of DNA and PDNA polymer distribution after polymerization for 10 min of the four reactant tiles at ∼1 μM
each and quenching with ∼1.5 μM terminator tiles (section 6 of the
Supporting Information). (B and C) Scans of the experimental gel
proﬁles (gray lines) and their deconvolution (black lines) for DNA and
PDNA, respectively. (D) Plot of the dsDNA equivalent size of PDNA(2) and DNA (0) polymers vs the number of units. Slopes correspondto 67 and 40.5 bp/unit for PDNA and DNA, respectively.
(E) Quantiﬁcation as equivalent masses of bands from panels B(white bars) and C (black bars) as a function of polymer size. The error
in band quantiﬁcation depends on methods but does not exceed arelative error of 1015%.

calculated as the sum of the real size of the nucleic part and of acontribution of the protein equivalent to 10 dsDNA bases perprotein unit. This value was set to be identical to the protein
contribution evidenced in gel filtration experiments. The figure
illustrates that experimental courses were well-simulated withthis same model for DNA- and PDNA-based experiments. This
indicates that, for large linear assemblies, no significant additional
factor, which could differentiate PDNA from DNA, needs to betaken into account to describe the global behavior.
For further comparison, the size distribution of polymer
species was analyzed by gel electrophoresis and proﬁles were
deconvoluted as previously described21 to calculate the electrophoretic mobility and for quantiﬁcation (Figure 8AC). However, in this case, terminator tiles (namely, am, Ma0 , bm, andMb0 ) able to cap the free ssDNA parts (A, A0 , B, and B0 ,respectively) of polymer extremities have to be added to quench
the polymerization and allow gel electrophoresis analysis. The
quenching process was incorporated into the simulation model(sections 5 and 6 of the Supporting Information).21 For DNA
and PDNA polymers, the apparent size was found to be proportional to the number of repeats with an additional contribution of
single-stranded quencher ends (Figure 8D). As expected, the size
step between bands appeared to be signiﬁcantly larger for PDNA(∼65 bp equivalent) than for DNA (40 bp equivalent) becauseof the contribution of protein domains to PDNA. However,relative distributions of polymer concentration obtained inDNADNA and PDNADNA experiments were found to be
similar (Figure 8E) and consistent with simulations. At this point,
we can conclude that DNA and PDNA recognition in solution is
fully driven by the nucleic acid part with almost undetectable

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Figure 9. SPR analysis of the interaction of PDNA assemblies and of
their b5 component with phospholipids. (A) Binding kinetics of the b5
component of PDNAs in the monomeric state (left) or following protein
dimerization through a disulﬁde bond (right). The W label denotes thestart of the washing phase. (B) Step by step self-assembly of PDNAs on amembrane surface. Free PDNA (1), supported membrane-bound
PDNA (2), free DNAtile dimer (3), coupling product of steps 2 and3 (4), free hybrid (5) between one PDNA and two DNA tiles, andcoupling product of steps 4 and 5 (6). (C) SPR sensorgram corresponding to panel B. The numbers correspond to steps depicted in panel B.
The diﬀerential reﬂectivity for each step is indicated by the arrows.

interference of the protein domain, except for hydrodynamic
eﬀects associated with size diﬀerences.
Self-Assembly of PDNAs on Vesicles and Supported
Membranes. The protein domain of PDNA carries a C-terminal
His4 tag that can form a complex with nickel, cobalt, or copper
divalent ions, mediating association through a surface and thus
constraining self-assembly mechanisms. While the affinity ofshort His tag sequences for the nickel-nitriloacetate linker issufficient for tight binding on affinity columns, the lifetime ofsuch a complex is fairly short, generally a few seconds.14,3032
Binding of the PDNA protein part to the phospholipid bilayer
supplemented with DOGS-Ni was evaluated using 100 nm
calibrated liposomes. The protein and phospholipid saturation
curves are illustrated in section 7 of the Supporting Information.
Half-saturation for the monomeric b5 domain used in PDNA
construction was found to be ∼5 μM, and the binding capacity
was clearly proportional to the total phospholipid concentration,
leading to an average binding surface of ∼50 nm2 for a single
b5 unit.
Assembly of PDNA on a membrane was further investigatedusing a DOGS-NTA-Ni-doped hemimembrane supported ongold, which allows monitoring of assembly by surface plasmon
resonance (SPR). Figure 9A illustrates that the binding of theprotein part of PDNA to the supported membrane is eﬃcient but
rapidly reversible with an aﬃnity in the micromolar range,
conﬁrming the previous observation with vesicles. However,when the protein parts form a dimeric structure, a stable association is observed with a dissociation rate hardly measurable (hour1829dx.doi.org/10.1021/bc2002264 |Bioconjugate Chem. 2011, 22, 1824–1834

Bioconjugate Chemistry

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Figure 10. High-speed AFM analysis of the dynamics of PDNA structures. (a and b) Dynamic AFM imaging (10 frames/s) evidencing the formation oftransient concatemers (species 1 and 2 giving 3 and 4 and 5 giving 6) of the construct described in the scheme. (c) Dynamics of two constructs anchoredonto a nanoparticle. Images from lane a were untreated, while nonrelevant background noise was removed for the sake of readability in lanes b and c.

range). This is due to a rebinding process in the diﬀusion limit
layer immediately surrounding the surface when at least two
linkages exist between the object and the surface. Such a situation
typically occurs when two PDNA tiles become linked throughhybridization with a cDNA tile. This principle is used in theexperiment described in Figure 9B and is illustrated by the SPR
sensorgram in Figure 9C. Formation of a supported membrane
hemilayer is evidenced by the fusion of DOGS-Ni-supplemented
phospholipid vesicles onto alkane thiol-modiﬁed gold. Binding of
PDNA to the Ni-NTA lipid head (steps 1 and 2) appeared to beeﬃcient, and the trace clearly illustrates that the bound PDNA
can be easily hybridized (steps 24) with the complementary
extremity of a DNADNA half-hybrid that acts as a linker
between two PDNAs (steps 46) ﬁnally stabilizing the wholeconstruct on the surface. However, the addition of mass following hybridization appeared to be signiﬁcantly below the expected
stoichiometry, and the defect increased with a larger number ofassembly steps. This could be explained by the fact that bindingof additional PDNA tiles to the surface is sterically impaired bytiles already associated on the surface. Reducing the eﬀect would
need careful control of surface saturation during assembly
initiation. However, this would suppose working under conditions far from surface saturation, which would in turn dramatically reduce the magnitude of the SPR signal, making multiplestep assemblies diﬃcult to monitor.
AFM Analysis of the Dynamics and Interactions of Short
PDNA Assemblies. A construct encompassing two PDNA tiles(scheme in Figure 10) was prepared and adsorbed onto freshly
cleaved mica in the presence of magnesium. High-speed AFM
imaging evidenced lateral mobility with large changes in position(tens of a nanometer) and shapes of objects within the 100 ms
time lapse between two scans (Figure 10a). An overlay ofconsecutive images separated by a 100 ms time lapse on a
300 nm  300 nm scale illustrates the presence in the populationof lower-mobility segments, while other regions of the molecules
were rapidly laterally diffusing (image sequence available insection 9 of the Supporting Information). Surface defects, suchas nanoparticles a few nanometers in diameter, can very efficiently trap diffusing PDNA structures, anchoring them onto a

Figure 11. Structures of immobilized PDNA assemblies. (A) Highspeed AFM imaging (raw data) of two assemblies and a schematic
description of the image. (B) Three other objects are illustrated at ahigher resolution (raw data), and section proﬁles of the DNA (1) andprotein (2) parts are given. Constructs encompassing ﬁve PDNA units
hybridized to a large single-stranded DNA were prepared as outlined in
the right part of the scheme (see section 8 of the Supporting Information
for details).

specific point of the surface when the remaining parts are still
stochastically moving around leading to tweezerslike structures(Figure 10a, lane 3). Interestingly, two or more constructs canfrequently interact together, resulting in the apparent fusion of
objects to give unstable longer species (Figure 10a, lane 2). The
events appeared to be mostly conservative with respect to length1830

dx.doi.org/10.1021/bc2002264 |Bioconjugate Chem. 2011, 22, 1824–1834

Bioconjugate Chemistry(daughter species has a length close to the sum of the parents), a
feature expected for classical base pairing. However, the construct exhibits single-stranded extremities that are not complementary and cannot give rise to a stable duplex. Consistently, thespecies appeared to be monodisperse in gel electrophoresis.
However, formation during gel filtration of shoulder peaks withmolecular masses greater than that of the main species suggested
weak interactions of construct extremities, consistently withpredicted ΔG values of 7.8 (b/b), 7.5 (a0 /a0 ), and 3.6
kcal/mol (b/a0 ) for the formation of intermolecular hydrogenbonds in a salty solution (section 10 of the Supporting Information). Such energies are sufficient to create short life (fewseconds) but easily observable concatemers under dynamic AFM
conditions. Statistical analysis of the length distribution of theconcatemer (one to eight repeats) was performed on 106 objects,
and the resulting profiles were found to be consistent with amodel in which single-stranded extremities of the species interact
together on the surface with ΔG values of 1.8 and 0.8 kcal/
mol, respectively (section 11 of the Supporting Information).
The observation was found to be dentical upon imaging of
DNA and PDNA of identical nucleotidic structures. As a control,
concatenation events were never observed with a dsDNA(513 bp) that does not include single-stranded extremities.
Structural Analysis of Large PDNA Structures. It could
appear surprising that the protein domains were hardly visible
under previously used experimental conditions. However, thelinker attaching DNA and protein domains in PDNA is sufficiently flexible to allow fairly different dynamics for the twocomponents. Under previously used conditions, the scanning
rate probably remained too slow compared to the dynamics ofthe protein domain to capture the 2 nm globular structuraldetails. To limit the overall dynamics, larger objects were
designed, including a 513-base single-stranded DNA scaffold
on the middle of which up to five PDNA tiles can be hybridized(Figure 11A). As previously reported,33 ssDNA segments generate tightly folded regions strongly interacting with the substrate. Under such conditions, a string of protein domains(thickness of 2.3 nm) regularly placed (1015 nm) along the
DNA filament (thickness of 1.35 nm) can clearly be imaged byhigh-speed AFM. Control involving dsDNA with same sequence
illustrates a filament with a homogeneous thickness of ∼1.3 nm(not shown). This illustrates that PDNA building blocks can beeasily used to decorate a DNA scaffold.

’ DISCUSSION
Covalent coupling of protein and DNA domains into a single
building block is a versatile alternative to noncovalent complexes
for building proteonucleic structures.3437 Diﬀerent PDNAs
encompassing a cytochrome b5 protein domain linked to a
ssDNA were designed, synthesized, and puriﬁed to homogeneity.
The C-terminal His tag on the protein part oﬀers a way to create
more speciﬁc and localized interactions with substrates thanDNA alone that nonspeciﬁcally interacts with ion-doped material
like Ni2+-treated mica.33 The requirement to build PDNAs is theavailability of a unique grafting position in the protein sequence.
Use of a surface cysteine residue is a method of choice, butalternate solutions include the use of a rare or non-natural aminoacid like a selenocysteine or an N-acetylphenylalanine,38 which
can be selectively grafted using adapted chemistry. A genericapproach could also involve grafting of the surface cysteine
opposite the CDR3 binding region onto small (14 kDa) VHH

ARTICLE

single-chain antibodies. Such antibodies could be used in turn toform a noncovalent high-aﬃnity complex with any protein ofinterest.39
This work speciﬁcally aimed to compare the physicochemicaland self-assembly properties of DNA and PDNA in diﬀerent
environments. Deﬁning and controlling parameters implied inself-assembly mechanisms of such hybrid synthetic structures will
permit us not only to use them as modular building blocks for
multifunctional nanostructures but also to improve our understanding of properties of natural nucleoproteic complexes.
A contribution of the protein domain to the hydrodynamic
properties of PDNAs was clearly evidenced by gel ﬁltrationand electrophoresis in SDSPAGE and nondenaturing agarose
gels. In all cases, the protein contribution appeared to be additive
with respect to the one of the nucleic acid parts provided to use
an equivalent DNA length that diﬀers depending on the analysis
method considered. During gel ﬁltration, the 13 kDa protein
domain migrates like a solvated dsDNA of 6.6 kDa. This contrasts with an equivalent mass of 1617 kDa observed in nondenaturing agarose gel electrophoresis calibrated using dsDNA.
This last value is consistent with a mass of 15 kDa for the protein
domain in SDSPAGE following calibration with proteins. This
is in agreement with a common extended conformation of thereference molecules (DNA and protein) used for calibration inan agarose gel and during SDSPAGE, respectively. During
HPLC gel ﬁltration, hydrodynamic constraints resulting fromﬂow are expected to favor a rather extended conformation of the
DNA used for size calibration. In contrast, the protein domain
remains globular, which tends to decrease its relative apparent
size. Reciprocally, during agarose gel electrophoresis, the much
lower charge of the protein domain compared to that of DNA
decreases the relative protein mobility, thus compensating for the
more compact structure of the protein domain. The strictly
additive contributions of double-stranded nucleic and protein
domains to hydrodynamic behaviors for a variety of PDNA
assemblies suggested that conformational interactions between
the two components are likely weak under all tested conditions
and do not aﬀect their individual shapes.
In contrast to pure nucleic structures that are generally
assembled under conditions close to the melting temperature
to avoid formation of dead-end parasitic hybrids,6,20 proteo
nucleic structures must be preferentially assembled in a kinetically controlled manner,4042 far below the melting temperature
to prevent thermal denaturation of protein domains. Hybridization rates are thus critical parameters of PDNAs that wereanalyzed by monitoring hypochromic eﬀects associated withhybridization in rapid-mixing experiments. While the length of
hybridizing segments is homogeneous for all considered tiles,
observed hybridization rates signiﬁcantly diﬀer over a 4-foldrange. This dispersion does not simply correlate with obvious
sequence features and for the same hybridizing segment depends
on the nature of ﬂanking regions. The reason is likely related to
the formation of possible secondary structures of ssDNA parts,
some of them having predicted stability suﬃciently large (8
14 kcal/mol) to signiﬁcantly interfere. In contrast, comparison of
the hybridization rates of DNA and corresponding PDNA does notevidence a signiﬁcant eﬀect of the protein domain when grafted on
the oligonucleotide 30 -end. However, a moderate decrease in
hybridization rates was observed for internal grafting proximal tothe hybridizing segment. In this case, base modiﬁcation by the linker
and protein presence exhibited cumulative eﬀects. When hybridization between larger polymers was considered, no additional eﬀect1831

dx.doi.org/10.1021/bc2002264 |Bioconjugate Chem. 2011, 22, 1824–1834

Bioconjugate Chemistry
was observed compared to the situation with short building blocks,
suggesting the absence of long-range interaction between PDNAs
when formation of linear polymer was considered.
Altogether, our results indicate that DNA and PDNA tiles aremostly equivalent at the level of their hybridization properties
and that PDNA can easily substitute for DNA in most DNAbased supramolecular assemblies, provided that the folding cansterically accommodate the protein parts. A potential limitation
remains the thermal stability of the protein domain under the
fairly high-temperature conditions required to prevent formationof a dead-end product by spurious hybridization.6,43 However,analysis of the thermal stability of our constructs evidenced thatlinkage to the nucleic acid part has no signiﬁcant detrimental
inﬂuence on protein stability leading, in our case, to constructs
stable up to 55 °C, a value that is compatible with current
hybridization procedures. Thus, any reasonably stable protein
can be substituted for our cytochrome b5 domain, including
recognition protein-like single-chain antibodies of the VHH
family. In the case of very unstable protein domains, an alternative assembly procedure under kinetic control remains
possible using, for example, DNA hairpin or loop motifs asprogrammable units.41,44 A combination of kinetic and thermodynamic control can also be used to direct self-assembly processes by engineering intra- and interparticle DNA hybridization.5,45
PDNAs potentially oﬀer many advantages when self-assemblies on surfaces are targeted. The protein domain can be used,
for example, for the recognition of surface chemical or physicalfeatures using material speciﬁc aﬃnity domains.46 This also
applies to redox active proteins (like cytochrome b5), for whichelectron transfer properties at surface interfaces can be modulated by the conformation of the DNA scaﬀold, thus constituting
biosensors. Analysis by rapid-scan AFM clearly evidenced lateral
diﬀusion properties of PDNA assemblies on surfaces. SPR
experiments demonstrated that such structures can be sequentially built by stepwise extension from a matrix-bound primer19
or by self-organization of tiles attached to membranes (thiswork). However, the progressive yield decrease during constructions resulted either from surface saturation or, as shown by HSAFM, from the presence of surface defects able to anchor laterally
diﬀusing PDNA molecules. A possible turnaround to build
defect-free surfaces would be the use of phospholipid vesicles
as assembly frame. This work illustrates that the fairly limited
stability of the attachment of PDNA to NTA-modiﬁed phospholipids is a limitation, which could be somewhat overcome whenseveral protein modules are bound to the same vesicles. Alternatively, multiple-head NTA constructs can be used to increaseaﬃnity.13,14,3032 However, the fairly rigid structure of large
PDNADNA supramolecular assemblies was found to conﬂictwith the strong curvature of small (50100 nm) vesicles andmore importantly can reciprocally induce an unwanted phasetransition of the support phospholipid structures, leading touncontrolled organization.
Formation of transient end-to-end concatemers was evidenced by HS-AFM upon lateral diﬀusion of PDNA assemblies
on the surface. Similar behavior was observed when DNA wassubstituted with PDNA under the same experimental conditions.
The phenomenon was clearly related to the presence of ssDNA
extensions on constructs that can form short stretches of paired
bases (four to six bases) and was not observed with fully dsDNA.
Interaction energies deduced from modeling of the concatemer
distribution appeared to be ∼4-fold smaller than calculated

ARTICLEvalues in solution, which is consistent with the much lower salt
concentration used under AFM imaging conditions. Formationof these transient structures while they are reversible competes
with true hybrid formation during assemblies slowing the process
on surfaces. The phenomena could be enhanced by the presenceof surface defects that eﬃciently trap diﬀusing PDNA assemblies
and by the observed topological cluttering on the surface (AFM
and SPR experiments). These three eﬀects combine in reducing
the self-assembly eﬃciency of surface-bound objects.
While high-speed AFM opens a new dimension for the
analysis at the single-molecule level of nucleic acid assemblies,
diﬃculties could arise from the relative values of scan rate andobject dynamics. Our data evidence that the small b5 domain(13 kDa) was not visible when attached to highly dynamic
structures, whereas it was clearly imaged when bound to muchlarger DNA exhibiting a lower mobility. Thus, it seems that somecompromise has to be found between resolution and dynamic
analyses, particularly when very small objects are involved. While
dynamic AFM observation of a fairly small protein (calmodulin,
17 kDa) bound to a streptavidin crystal was recently reported,47
our results go further than previous reports48 by visualizing forthe ﬁrst time a small protein domain (13 kDa) linked to isolated
DNA molecules in solution.

’ ASSOCIATED CONTENTbS Supporting Information. DNA tile sequences andmatches (section 1), schematic for the synthesis of proteinDNA (PDNA) structures (section 2), synthesis of PDNA structures (section 3), contribution of building blocks to the apparent
size in gel ﬁltration and electrophoresis (section 4), experimental
and simulated proﬁles for PDNA copolymers (section 5), parameter sets for simulations (section 6), cytochrome b5 and phospholipid concentration dependence for the formation of proteinlipid complexes (section 7), puriﬁcation of the DNA and
PDNA assemblies described here (section 8), high-speed AFM
image sequences (section 9), thermodynamic parameters(section 10), and statistical analysis of the formation of concatemers (section 11). This material is available free of charge via
the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION
Corresponding Author*Telephone: +33 567048806. Fax: +33 561559400. E-mail:denis.pompon@insa-toulouse.fr. Present address: LISBP/INSA,
UMR INSA/CNRS 5504, 135 Avenue de Rangueil, 31077
Toulouse cedex 4, France.
Funding Sources

We acknowledge the ﬁnancial support of this work by Grants
ANR-05-NANO-021-04, ANR-08-NANO-010-01, and ANR08-PCV08_343399 from the Programme Nanosciences et Nanotechnologies (PNANO) and Programme interdisciplinaire en
physique et chimie du vivant (PCV) actions of the National
Research Agency (ANR), France.

’ ACKNOWLEDGMENT
We thank Dr. Takayuki Uchihashi (Physics Laboratory,
Kanazawa University), Drs. Ignacio Casuso and Simon Sheuring(Institute Curie, Paris, France), Dr. Pierre-Emmanuel Milhiet
1832

dx.doi.org/10.1021/bc2002264 |Bioconjugate Chem. 2011, 22, 1824–1834

Bioconjugate Chemistry(CBS, Montpellier, France) for technical support, and Dr. Gilles
Truan for proofreading the manuscript.

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