Reversible Hybridization of DNA Anchored to a Lipid Membrane via

Article pubs.acs.org/Langmuir

Reversible Hybridization of DNA Anchored to a Lipid Membrane via Porphyrin Jakob G. Woller,† Karl Börjesson,† Sofia Svedhem,‡ and Bo Albinsson*,† †

Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, S-41296 Gothenburg, Sweden ‡ Department of Applied Physics/Biological Physics, Chalmers University of Technology, S-41296, Gothenburg, Sweden S Supporting Information *

ABSTRACT: The binding of zinc−porphyrin-anchored linear DNA to supported lipid membranes was studied using quartz crystal microbalance with dissipation monitoring (QCM-D). The hydrophobic anchor is positioned at the ninth base of 39base-pair-long DNA sequences, ensuring that the DNA is positioned parallel to the membrane surface when bound, an important prerequisite for using this type of construct for the creation of two-dimensional (2D) DNA patterns on the surface. The anchor consists of a porphyrin group linked to the DNA via two or three phenylethynylene moieties. Doublestranded DNA where one of the strands was modified with either of these anchors displayed irreversible binding, although binding to the membrane was faster for the derivatives with the short anchor. The binding and subsequent hybridization of single-stranded constructs on the surface was demonstrated at 60 °C, for both anchors, revealing a coverage-dependent behavior. At low coverage, hybridization results in an increase in mass (as measured by QCM-D) by a factor of ∼1.5, accompanied by a slight increase in the rigidity of the DNA layer. At high coverage, hybridization expels molecules from the membrane, associated with an initial increase, followed by a decrease in DNA mass (as detected both by QCM-D and by an optical technique). Melting of the DNA on the surface was performed, followed by rehybridization of the single-stranded species left on the surface with their complementary strand, demonstrating the reversibility inherent in using DNA for the formation of membrane-confined nanopatterns.

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INTRODUCTION DNA nanotechnology has grown tremendously during the past decade.1 This is motivated by the unique self-assembly and programmable properties provided by the specific Watson− Crick base pairing of the DNA molecule. In this way, various one-, two-, and three-dimensional objects have been created and visualized by microscopic techniques, such as atomic force microscopy (AFM)2 and electron microscopy.3 The development of techniques to synthesize branched DNA oligomers4−6 and utilization of naturally occurring polynucleic acids7 has led to a formidable explosion of available structures, ranging from small objects of 5−10 nm to large complexes of 100−200 nm, all in principle addressable down to a single base pair resolution. This technology is now ripe for moving toward applications. However, for this to happen, ways to functionalize the DNA nanostructures need to be developed. In addition, to harvest the full potential of the self-assembly properties of DNA, it is of importance to have structures that assemble and reassemble in fluid solution (water). At the same time, for many potential applications, the DNA nanoconstructs must be attached to surfaces. We propose to meet these seemingly contradictory demands through the assembly of DNA nanoconstructs anchored with lipophilic porphyrins to a lipid membrane. The surface of a lipid membrane provides the © 2011 American Chemical Society

perfect environment for DNA components to diffuse and hybridize. It furthermore provides a two-phase system: one aqueous phase where the DNA nanostructure resides, and one lipophilic phase where the redox active porphyrins could be used to initiate chemical reactions. In this way, we have previously demonstrated how linear8 and hexagonal9 DNAs modified by lipophilic porphyrin molecules were anchored to liposomes, resulting in the DNA being in the water phase and the porphyrin anchor in the membrane. For the prospect of using these nanostructures for applications, it should be noted that the porphyrin is not merely an anchor, but can, upon photoexcitation, also function as an excellent electron donor.8,10 Previous studies on amphiphilic DNA−porphyrin systems anchored to liposomes with porphyrin-modified nucleotides have been performed in bulk solution using spectroscopic techniques, and have focused on probing the environment of the porphyrin. Here we move toward supported membranes as Special Issue: Bioinspired Assemblies and Interfaces Received: October 12, 2011 Revised: December 21, 2011 Published: December 27, 2011 1944

dx.doi.org/10.1021/la2039976 | Langmuir 2012, 28, 1944−1953

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Figure 1. Schematic representation of the major binding and hybridization events described in this study. Reaction arrows show the direction in which the equilibrium is shifted at room temperature. The left panel shows that the binding of single-stranded porphyrin−DNA is strong, although the porphyrin−DNA is shifted toward a form that cannot bind to the membrane. By increasing the temperature to 60 °C, binding is greatly enhanced. The right panel shows strong binding of double-stranded porphyrin−DNA. The middle panel shows that addition of complementary single strand induces hybridization on the membrane surface, which is reversed by heating to 80 °C. Inset: The two porphyrin-modified nucleosides used in this study denoted by the length of the hydrophobic connecting linker between DNA and porphyrin; bi-pe (two phenylethynylene moieties) or tri-pe (three phenylethynylene moieties).

tri-pe, as shown in Figure 1, depending on whether two- or three-unit phenylethynylene linkers are used (bi-pe and tri-pe, respectively) for the two modified nucleosides (the molecular structures are shown in the figure inset). The QCM-D data are complemented by dual polarization interferometry (DPI) and fluorescence recovery after photobleaching (FRAP) measurements to give a more complete picture of the binding of the DNA constructs to the membrane, as well as their lateral diffusion when confined to the membrane. The aim of the present study was to characterize, by means of surface-sensitive analytical techniques, the insertion of porphyrin-modified DNA strands into supported lipid membranes. To ensure that our anchoring strategy allows the DNA to retain its properties of specific base pairing, a necessary prerequisite for using these constructs for the formation of functional nanoarchitechtures, a further goal was to show that reversible hybridization was possible at the membrane surface. Studies of amphiphilic DNA constructs at supported lipid membrane surfaces have been described previously, where the DNA strands have been modified at either the 5′ or 3′ end by various lipophilic anchors. Phospholipid anchoring of DNA has been used to sort labeled vesicles12 and to dock vesicles

the substrate for anchoring the DNA molecules, which is much more relevant from a technological perspective. This, however, requires surface-sensitive techniques to characterize and quantify the binding of these dynamical systems. Quartz crystal microbalance with dissipation monitoring (QCM-D) has proven to be an excellent surface analytical technique for the study of thin hydrated films. Although, in general, the sensitivity of acoustic sensor techniques is lower than for surface plasmon resonance (SPR), or interferometry, the fact that QCM-D measures the acoustically coupled mass, instead of the biomolecular mass, increases the sensitivity compared to these other surface techniques for molecular films with a high water content.11 The QCM-D technique, in addition to measuring the acoustically coupled mass, also measures the energy dissipation, which is related to the rigidity of the layer probed, allowing insight into the structural properties of the DNA film formed at the membrane surface. We have used QCM-D to study the binding of porphyrin-modified DNA strands to lipid membranes, varying between single- (ss) and double-stranded (ds) constructs, as well as between a shorter and a longer linker between the porphyrin and the DNA. The DNA samples are designated ss bi-pe, ss tri-pe, ds bi-pe, and ds 1945


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together at the membrane surface.13 Similar studies have been performed using cholesterol-anchored DNA,14 where two cholesterols allowed for irreversible binding of DNA strands to the membrane,15 and the study of fusion of liposomes with supported membranes.16 Multicholesterol anchoring has also been demonstrated, showing reversible hybridization at the surface.17 Modifying DNA with biotin is an alternative method for binding of DNA to membranes, although the binding is mediated by streptavidin attached to the membrane surface.18,19 The DNA constructs used in this study are original in their design compared to the previous studies with respect to the built in functionality of the hydrophobic anchor, and also by the placement of the porphyrin group away from the end of the molecule.

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spectroscopically well-characterized system and that absorption spectra were identical to those obtained in previous studies.8,10 A linear combination of the nucleotide absorption coefficients at 260 nm was used to set unmodified DNA strand concentrations (εss comp = 361 600 M−1cm−1, εss non‑comp = 375 300 M−1cm−1). For samples containing porphyrin-modified nucleotides (both double and single strands), concentrations were set using porphyrin Q-band absorption at 545 nm (εbi‑pe/tri‑pe = 14 000 M−1cm−1). QCM-D Experiments. QCM-D-data, in the form of frequency shifts (Δf) and dissipation shifts (ΔD) were recorded at room temperature (22 °C) on an E4 instrument (Q-Sense, Sweden). For measurements at elevated temperatures, a QHTC 101 temperature control chamber (Q-Sense, Sweden) was used, and the temperature was set to 60 °C, unless otherwise stated. Sensors used were SiO2 sputter-coated AT-cut quartz crystals with a fundamental frequency of 5 MHz (Q-Sense, Sweden). The sensors were stored in a 10 mM sodium dodecyl sulfate (SDS) solution between measurements, and prior to measurements they were thoroughly rinsed with water, dried in N2 flow, followed by 45 min of UV/ozone treatment, and then mounted in the flow cell chamber. The mounted sensor was left in water until a stable baseline was obtained (less than 1 Hz/h drift). The buffer was degassed prior to measurements. For measurements at elevated temperatures, samples and buffer were heated to 2 °C above the chamber temperature, prior to injection. Measurements at 60 °C often resulted in the formation of air bubbles, which were clearly seen to shift the frequency signal to less negative values, whereas the dissipation signal was less susceptible to disruption. A constant flow rate of 50 μL/min was used unless otherwise stated. Frequency and dissipation shifts for odd overtones (3rd to 13th) were collected. Frequency shifts were normalized to the fundamental frequency by division by the overtone number. Supported lipid membranes were formed in situ by flowing a 0.1 mg/mL solution of DOPC liposomes over the sensor surface, as described previously,20,21 and resulted in frequency shifts of Δf ≈ −26 Hz (corresponding to a mass of 460 ng/ cm2) and a dissipation shift of ΔD < 0.5 × 10−6. After completed measurements, 10 mM SDS was flowed over the surface, removing both lipids and porphyrin−DNA. Surfaces were then rinsed with water, and finally buffer was again added to reach frequency and dissipation shifts equal to those obtained at the start of the measurement, indicating that no porphyrin−DNA had irreversibly attached to the surface. Following cleaning of the sensor in the chamber, it was possible to form membranes again by addition of new liposomes. For melting experiments, a saturated layer of ds tri-pe was formed, after which the flow was stopped and the chamber was heated to 80 °C. After stabilization, buffer (preheated to 82 °C) was flowed over the surface for 15 min. The flow was stopped, and the chamber was cooled to room temperature and left to stabilize. Once stabilized, the complementary strand, ss comp, was added. In these experiments, the QCM-D curves at room temperature obtained during the last part of the experiment were shifted such that the level for the clean surface corresponded to the level for the clean surface before the temperature treatment. The initial binding rate (kinit) was used to quantify kinetics from QCM-D frequency data. It was defined as the slope of the frequency shift versus time for the first 5 min of binding, and was obtained through linear regression. The estimated uncertainty in kinit, based on two measurements of ds tri-pe, is 10%. To quantify the rigidity of the layers formed, the value of −ΔD/Δf was used. The rigidity is related to the effective viscosity obtained when performing viscoelastic modeling of the data.19,22,23 A rigid layer has a low value of −ΔD/Δf, and a high modeled effective viscosity. A soft layer has a high value of −ΔD/Δf and a low modeled effective viscosity. To quantify the coveragedependent contribution of porphyrin−DNA to the softness/rigidity of the layer, a linear regression of dissipation versus frequency shift data was performed, defined as −df/dD, resulting in an uncertainty of 10% based on seven measurements of ds tri-pe at concentrations between 10 and 100 nM. The Sauerbrey model was used to relate frequency shifts to mass changes.24

MATERIALS AND METHODS

The buffer used for all experiments was 25 mM Tris containing 200 mM NaCl, adjusted to pH 8.0 using HCl. Water was purified using a Milli-Q water purification system (Millipore, U.S.). Liposome Preparation. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) dissolved in chloroform was obtained from Avanti Polar Lipids, U.S. Chloroform was removed from the solution using a rotavapor, followed by further drying at higher vacuum for >4 h. The dried lipid film was rehydrated in buffer, followed by five cycles of freezing in liquid N2 and thawing at 35 °C, after which the suspension was extruded 21 times through polycarbonate filters (Avestin, Canada, pore size 100 nm diameter). The final liposome concentration was ∼4 mg/mL. Mean liposome diameters were determined by dynamic light scattering using a Zetasizer Nano zs (Malvern, U.K.) at a lipid concentration of 0.016 mg/mL to be ∼120 nm, with a polydispersity index of 24 h, rinsed thoroughly with water, dried under N2 flow, and treated with UVozone for 45 min. Cleaned coverslips were mounted in an in-house made flow cell. Buffer and subsequently 0.1 mg/mL DOPC liposomes in buffer were flowed over the surface and left to stand for 0.5−1 h for formation of a supported lipid membrane. After membrane formation,

Figure 2. QCM-D frequency (A) and dissipation (B) shifts obtained for the binding of 50 nM ds tripe to a supported DOPC lipid membrane under 50 μL/min flow.

QCM-D frequency and dissipation shifts (Δf and ΔD, respectively) obtained upon flowing a 50 nM solution of ds tri-pe over a DOPC supported lipid membrane, which had been formed on the sensor surface just prior to addition of the porphyrin−DNA construct. As expected, based on previous 1947


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spectroscopic studies,8,10 accumulation of the porphyrinmodified DNA at the membrane is observed. The mass uptake is evidenced by a monotonic frequency decrease. The frequency curve follows a monoexponential form versus time until reaching approximately −22 Hz for all overtones, after which the binding continues with a much slower rate. The slow binding observed at high coverage may be due to porphyrin− porphyrin or DNA−DNA interactions, resulting in a reordering on the membrane as the concentration increases, thereby allowing more porphyrin−DNA to bind. The dissipation increases in a monotonic manner, similar to that of the frequency, and reaches a maximum value of ΔD = 2.0 × 10−6. The value of −ΔD/Δf, which is related to the rigidity of the film, is 9.1 × 10−8 Hz−1. The relatively small dissipation shift compared to the frequency shift is characteristic for a fairly rigid adsorbed layer. For such layers, the Sauerbrey equation (eq 1) is a good model, according to which the mass uptake at the surface is proportional to the corresponding frequency shift. Using this relation, the saturated amount of ds tri-pe at the membrane surface corresponds to an acoustically coupled mass (ΔmQCM) of 420 ng/cm2. This mass is about 3 times larger than the maximum theoretical mass of a saturated layer of ds tri-pe (see Supporting Information). This discrepancy is expected, and it is due to the water content of the DNA film, as the QCM-D technique is sensitive to all mass that is acoustically coupled to the sensor surface, i.e. the DNA mass as well as the associated water content. Hydration and the effect it will have on the area occupied per molecule will be considered below. Upon rinsing of the ds tri-pe saturated membrane with buffer, only small frequency and dissipation shifts are observed (∼1 Hz and 0.1 × 10−6, respectively), indicating that the binding of ds tri-pe to the membrane is nearly irreversible under these experimental conditions. Furthermore, no measurable change in frequency (Δf < 0.5 Hz) is observed when buffer is flowed over a membrane with ds tri-pe at 65% coverage for 10 h, further strengthening the observation of irreversible binding. This tight binding and lack of desorption is similar to data obtained in previously published studies of double-cholesterol-anchored DNA (30 mer hybridized with 15 mer) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) supported membranes, in contrast to a single cholesterol at the end of DNA, which does not bind irreversibly.15 To test for possible concentration dependencies, binding of ds tri-pe to supported lipid membranes was studied at concentrations between 5 nM and 100 nM. Binding at 5 nM was not followed to saturation due to slow binding kinetics. For concentrations between 10 nM and 100 nM, both frequency and dissipation shifts reached similar saturation values. Using a Langmuir 1:1 binding model (eq 2), saturation at a given concentration will only reach the maximum saturation value if the dissociation equilibrium constant, KD, is much lower than the concentration used. Therefore, KD for ds tri-pe can be estimated to be much lower than 10 nM. This value should be compared to the KD obtained for binding of ds tri-pe to liposomes in bulk solution, which was found to be 120 nM.8 This shows that the binding to a supported membrane is at least an order of magnitude stronger than binding to a liposome for this construct. The large difference in KD between liposome and supported membrane may be due to a difference in membrane properties, where the larger curvature of the liposomes results in a more dynamic membrane, lowering the affinity for porphyrin−DNA compared to supported mem-

branes. The proximity of the supported membrane to the solid surface is also likely to decrease membrane dynamics, thereby increasing the affinity of porphyrin−DNA. Another possible explanation is that the two methods of measurement are very different, and therefore probe different processes. Since such a large difference was observed in the equilibrium dissociation constant of ds tri-pe to supported lipid membranes compared to liposomes, FRAP measurements were performed to investigate whether the construct diffused on the membrane surface (see Supporting Information). This resulted in a diffusion constant of ∼3 μm2 s−1, similar to results for diffusion of dye-labeled lipids,29 and slightly faster than diffusion of a 2D DNA hexagon consisting of 189 base-pairs anchored by one ds tri-pe.9 No fraction of immobile molecules was revealed in the analysis. Figure 3 shows a plot of the dissipation shift versus the frequency shift (ΔD−Δf plot), yielding further information on

Figure 3. Plot of dissipation shift (ΔD) as a function of frequency shift (Δf), upon binding of ds tri-pe to a DOPC membrane, for the 7th, 9th, 11th and 13th overtone, showing two linear regimes of binding: one at low (regime 1) and one at high (regime 2) coverage. Experiments at concentrations between 10 and 100 nM yielded plots identical to this figure.

the structural properties of the DNA layer formed at different coverages of ds tri-pe at the membrane surface. In such plots, a straight line is expected for binding events where the binding of each additional porphyrin−DNA molecule induces the same frequency and dissipation shift. Two linear regimes are observed in the plot: one at low coverage (regime 1: 0 Hz > Δf > −5 Hz) and one at high coverage (regime 2: −12 Hz > Δf > −22 Hz). Thus, at low coverage, the expected linear ΔD−Δf response for a process where each ds tri-pe molecule binds independently to the membrane is observed. Above a certain coverage (Δf < −12), a second linear regime is reached, where each additional molecule binding to the membrane induces a smaller dissipation shift than at low coverage, until saturation has been reached. The behavior in regime 1 is intuitively understood, and also explained by a phenomenological model proposed by Bingen et al.,30 to account for the acoustically coupled mass sensed using QCM-D. In the model, each molecule can bind freely to the membrane until a certain coverage is reached, after which hydration decreases due to the close proximity of neighboring molecules. Such a change in hydration could also affect the rigidity of the layer, resulting in a more rigid layer at high coverage, as is seen in Figure 3. In our case, the behavior in regime 2 is perhaps a bit unexpected, showing that additional DNA molecules continue to contribute in a similar manner to the QCM-D result as the density of the packing increases. 1948


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Table 2. Saturation Masses (ΔmQCM, ΔmDPI), Initial Binding Rates (kinit), and Slopes of Dissipation versus Frequency Shifts in Two Binding Regimes (−dD/df) for Binding of ss bi-pe, ds bi-pe, ss tri-pe and ds tri-pe to Lipid Membranes at 22 °C and at 60 °C 22 °C strand ss bi-pe ds bi-pe ss tri-pe ds tri-pe

60 °C

ΔmQCM ng/cm2

kinit Hz/s

−dD/df (10 /Hz) reg. 1

−dD/df (10 /Hz) reg. 2

ΔmQCM ng/cm2

ΔmDPI ng/cm2

kinit Hz/s

−dD/df (10−6/Hz) reg. 1

−dD/df (10−6/Hz) reg. 2

a

0.0012 0.020 0 0.011

0.18 0.11

a

460 370

>53 54

0.028 0.040 0.0015 0.016

0.24 0.13 0.23 0.12

0.11 0.094

350 0 420

−6

−6

0.062

a

a

a

b

0.12c

0.066c

>350

b

a

0.079

a

Value was inaccessible due to slow or no binding. bConstructs were not measured. cValues are based on eight independent measurements at varying concentration yielding a standard deviation of 0.01 Hz−1 × 10−6 for regime 1 and 0.007 Hz−1 × 10−6 for regime 2.

increasing the temperature from 22 to 80 °C, is consistent with an aggregated form being present at room temperature (see Supporting Information). This interpretation is consistent with the slower binding of ss tri-pe compared to ss bi-pe, where both the larger hydrophobic area and longer anchor length allows more interaction between the porphyrin and DNA bases. For a double-stranded construct, such an interaction between the porphyrin and DNA is not possible for two reasons: (i) the bases are hydrogen bonded (hybridized) and thus shielded within the duplex, and (ii) the large persistence length of double-stranded DNA does not allow it to bend enough to interact with the porphyrin. This is in agreement with the much larger binding rate of double-stranded constructs. Binding of ds bi-pe reaches saturation (ΔmQCM = 350 ng cm−2) at smaller frequency shifts than for ds tri-pe and no slow binding kinetics is observed at high coverage, in contrast to ds tri-pe. Perhaps the shorter and stiffer anchor for ds bi-pe does not allow porphyrin−porphyrin interaction in the membrane, lowering the total mass bound compared to ds tri-pe. Binding of ds bi-pe at 50 nM and 100 nM gave similar saturation masses (ΔmQCM) indicating that, as for ds tri-pe, the concentrations used were too high for a determination of the dissociation equilibrium constant (i.e., the dissociation equilibrium constant, KD, is much lower than 50 nM). For binding of ds bi-pe to liposomes in bulk a value of KD = 380 nM was obtained,8 again showing that binding to supported membranes is much stronger than to liposomes. Plotting the dissipation versus frequency shift for all constructs reveals a clear difference between single- and double-stranded binding, whereas the ΔD−Δf responses for ds bi-pe and ds tri-pe are indistinguishable (Table 2). The similarity in the ΔD−Δf plot for ds bi-pe and ds tri-pe is expected due to their similar mass and structure. Binding of ss bi-pe results in a softer layer at low coverage with a higher absolute value of −dD/df compared to the two double strands. Data at high coverage were not obtained for single-strand binding due to the slow binding kinetics. Interaction of ss bi-pe, ds bi-pe, ss tri-pe and ds tri-pe with Supported Membranes at 60 °C. In order to efficiently bind single-stranded porphyrin−DNA into the membrane for hybridization studies, measurements at 60 °C were performed. Saturation masses ΔmQCM (obtained from Sauerbrey analysis), initial binding rates, kinit, at 100 nM, and the slope in ΔD−Δf regimes, −dD/df, for the interaction of ss bi-pe, ds bi-pe, ss tri-pe, and ds tri-pe with supported membrane at 60 °C are collected in Table 2, and the underlying QCM-D graphs are shown in the Supporting Information. For comparison, we first investigated binding of the doublestranded constructs at 60 °C. The increase in temperature has

Plotting dissipation shifts versus frequency shifts for concentrations of ds tri-pe ranging from 10 to 100 nM revealed identical plots, indicating that the two binding regimes are independent of bulk concentration (see also Table 2). This means that the transition between the two binding regimes occurs at the same coverage, independent of the concentration, and is a property of the layer formed by ds tri-pe. Thus, there seems to be no binding of aggregates, or other concentration dependent phenomena, in corroboration with bulk results for binding of ds tri-pe to liposomes.8 Comparison between ss bi-pe, ds bi-pe, ss tri-pe, and ds tri-pe at Room Temperature. Our bulk liposome measurements previously showed that incorporation of ss bipe and ss tri-pe into liposomes proceeds very slowly at room temperature, and that temperatures of 40 and 60 °C were required for efficient binding of ss bi-pe and ss tri-pe, respectively.8 However, since such a tight binding was observed for ds tri-pe to supported lipid membranes compared to liposomes, binding of single strands was also investigated at room temperature. The binding characteristics of ss bi-pe, ds bi-pe, ss tri-pe, and ds tri-pe, interacting with DOPC membranes, are summarized in Table 2, where they are expressed as initial binding rate, kinit, mass at saturation, ΔmQCM, and slope of the ΔD−Δf curve in the two different regimes, −dD/df. QCM-D frequency, dissipation, and ΔD−Δf plots for all samples are shown in the Supporting Information. In all cases, the recorded dissipation shifts were small enough compared to the frequency shifts to allow the application of the Sauerbrey equation for estimations of the mass accumulated at the surface. Binding of single strands proceeds with a very slow binding rate, similar to the observation for the binding of these constructs to liposomes in bulk. For ss tri-pe, the single strands with long linker, no binding was observed at room temperature, and the binding of ss bi-pe was not followed to completion due to slow kinetics. As a consequence, a study of hybridization at room temperature was not feasible. The binding of double strands is more than an order of magnitude faster than single strands, with the short anchor (ds bi-pe) in turn binding a factor of 2 faster than the long anchor (ds tri-pe). Similarly, flowing buffer over a saturated layer of ds bi-pe resulted in a faster desorption rate than for ds tri-pe. The slow binding rate of single-strand porphyrin−DNA constructs compared to the double strands, as seen here and in our previous work with liposomes in bulk,8 is attributed to an intramolecular hydrophobic interaction between the anchor and DNA bases in solution, shielding the anchor from entering the membrane (Figure 1, left panel). A change in the porphyrin Soret band absorption at 380−450 nm for ss tri-pe, upon 1949


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When converting the dissipation and frequency data for ss bipe binding into a ΔD−Δf plot (Figure 4), it is seen that

little effect on the binding of ds bi-pe and ds tri-pe to the membrane. There is an increase in the initial binding rate by a factor of ∼2 compared to room temperature, but the saturation frequency and dissipation shifts remain similar. In comparison to room temperature, the desorption rate increases, with ds bipe desorbing faster than ds tri-pe, as was seen at room temperature. The two binding regimes observed at room temperature for ds bi-pe and ds tri-pe in the ΔD−Δf plot, and characterized by the slope −dD/df, are still present at 60 °C. In regime 1, the −dD/df value is unchanged at the higher temperature, and a small increase in slope is seen in regime 2 for both ds bi-pe and ds tri-pe binding. This indicates that the layer obtained at high coverages is less rigid (with a lower modeled effective viscosity) at 60 °C compared to room temperature. As expected, the increase in temperature has a pronounced effect on the binding rates of single strands to the membrane. The initial binding rate, kinit, is still very slow for ss tri-pe, on the order of that of the ss bi-pe binding to the membrane at room temperature. This is too slow for using this strand in hybridization experiments at full coverage. For the shorter anchor (ss bi-pe), the initial binding rate increased 20-fold, allowing for saturation of the membrane. The saturated layer formed upon ss bi-pe binding induces frequency and dissipation shifts of −25 Hz and 4 × 10−6, respectively, with a corresponding value of −ΔD/Δf = 16 × 10−8 Hz−1. The saturated layer of ss bi-pe is therefore much softer than for ds bi-pe (with a value of −ΔD/Δf = 9.1 × 10−8 Hz−1), although the acoustically coupled masses are similar. Assuming that the hydration of single and double strands is similar,19 and knowing that the molecular weight of ss bi-pe is half of ds bi-pe, one can estimate that ∼2 times as many anchored molecules of ss bi-pe have bound compared to ds bi-pe, based on the similar frequency shifts obtained. This indicates that the looser single strands are able to pack closer together than the hybridized structures on the membrane surface. Reversible Hybridization on the Membrane Surface. The elevated temperature allowed binding of ss bi-pe to saturation, and hybridization experiments could therefore be performed on a saturated layer of ss bi-pe. Investigating the hybridization of ss bi-pe at full coverage was particularly interesting, since it was estimated that there are twice as many anchored molecules in a saturated layer of ss bi-pe compared to a saturated layer of ds bi-pe. Hybridization was investigated using QCM-D, and performed by allowing the single strand construct to bind to the bilayer, followed by addition of the complementary strand, ss comp (middle panel in Figure 1). For comparison, hybridization at half coverage of ss bi-pe and ss tripe was also performed (see Supporting Information for QCMD frequency and dissipation results of ss bi-pe hybridization at half coverage). We present the hybridization at half coverage first, since this will aid in the interpretation of hybridization at full coverage. At half coverage of ss bi-pe, hybridization causes a large decrease in frequency (i.e., increase in acoustically coupled mass) of −7 Hz (from −13 to −20 Hz), accompanied by a decrease in dissipation (i.e., increase in layer rigidity) of −0.3 × 10−6. Hybridization of ss tri-pe at similar coverage revealed similar results (not shown). The decrease in frequency is consistent with the increase in mass expected when going from single to double strand. The slight decrease in dissipation is attributed to the DNA stiffening upon hybridization.

Figure 4. QCM-D dissipation versus frequency shifts for the binding of ds bi-pe and ss bi-pe to a DOPC lipid membrane, and subsequent hybridization at half and full coverage of ss bi-pe. Point 1 marks addition of ss comp and hybridization at 50% coverage of ss bi-pe, resulting in a layer identical to saturated ds bi-pe (point 3). Point 2 marks addition of ss comp at full coverage of ss bi-pe, resulting in point 4, where hybridization is complete. From point 4, desorption continues until point 3 is reached, the point of a saturated membrane of ds bi-pe.

hybridization at half coverage (start at point 1 in Figure 4) results in a layer with exactly the same Δf and ΔD as seen for a saturated layer of ds bi-pe (point 3 in Figure 4). This indicates that all strands have hybridized, and a surface consisting of only ds bi-pe is obtained. These data further corroborate the estimation that more anchored molecules are present in a saturated layer of ss bi-pe compared to ds bi-pe, since hybridization at half coverage of ss bi-pe formed a f ull layer of ds bi-pe. QCM-D frequency and dissipation shifts for hybridization at full coverage of ss bi-pe are shown in Figure 5. Upon addition of ss comp to a saturated layer of ss bi-pe, a complex kinetic behavior is seen (arrows in Figure 5 and point 2 in Figure 4). An initial decrease in frequency (i.e., accumulation of mass) of −2 Hz is observed, followed by a slower increase in frequency of 7 Hz (i.e., removal of mass). The dissipation decreases monotonically, both during the decrease and increase in frequency. We attribute the initial decrease in frequency to hybridization on the surface, which expels molecules from the membrane causing a subsequent increase in frequency. The data can be understood in more detail by viewing Figure 4, and considering that (i) hybridization causes a straight line in the ΔD−Δf plot corresponding to a decrease in frequency and decrease in dissipation, as was seen for hybridization at half coverage, and (ii) desorption causes a different straight line corresponding to an increase in frequency and decrease in dissipation. Directly after ss comp is added (point 2 in Figure 4) hybridization dominates, and a straight line with decreasing frequency and dissipation is observed. As hybridization continues, desorption increases until hybridization is complete at point 4 in Figure 4. Point 4 coincides with a point obtained if the ΔD−Δf plot for ds bi-pe binding is extended to higher coverage, thereby indicating that hybridization is complete, and that only ds bi-pe is present on the surface. From point 4 desorption continues, until the value for a saturated layer of ds bi-pe is obtained (point 3). Addition of a noncomplementary strand to the membrane consisting of ss bi-pe showed no change in frequency or dissipation. 1950


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constructs is known, the biomolecular mass can be related to the area occupied per molecule. For binding of the double strand, ds bi-pe, a biomolecular mass, ΔmDPI = 54 ng/cm2 was obtained using the de Feijter equation (eq 3), indicating that the degree of hydration is ∼85% in the DNA film. Previous studies on end-on biotinylated DNA bound in a perpendicular position to the membrane have yielded a hydration of 90%.19 For ss bi-pe, binding, the biomolecular mass is 53 ng/cm2. The similar masses obtained for the single and double strand clearly show that there are ∼2 times as many anchored entities in the singlestranded layer, compared to the double-stranded layer, since the molar weight of the single strand is ∼50% of the double strand. Upon addition of the complementary strand to the ss bi-pe layer, the mass increases rapidly to 72 ng/cm2 followed by a slower decrease, which is due to dissociation from the membrane. These results are qualitatively in agreement with the above interpretation of the QCM-D data. Converting the mass at saturation to a molecular area (see Supporting Information) results in a molecular area for ds bi-pe of ∼80 nm2. This value should be compared to the projected area of the DNA construct, which is 26 nm2 (see Supporting Information), consistent with the formation of a monolayer at the surface. For ss bi-pe, the molecular area becomes 40 nm2, showing a tighter packing of the single-stranded constructs compared to the double strands. A major advantage of soft DNA nanotechnology is the possibility of exchanging specific parts of an advanced system, for example a single strand in a complex DNA structure. To test whether it was possible to melt the DNA helices at the membrane surface, QCM-D measurements were performed at varying temperatures. For these measurements, ds tri-pe was investigated, since the desorption rate of this construct was observed to be slower than for ds bi-pe. ds tri-pe was allowed to bind to a DOPC membrane at room temperature, after which the complementary strand was removed by heating to 80 °C, followed by cooling to room temperature (Figure 6). Thus,

Figure 5. QCM-D frequency (A) and dissipation (B) shifts obtained for the binding of ds bi-pe and ss bi-pe to a DOPC membrane at 60 °C. Following saturation with ss bi-pe, ss non-comp was added, resulting in no change in frequency or dissipation. Hybridization using the complementary strand, ss comp, is observed.

To exemplify the effects of hybridization on DNA strands attached parallel to the surface, it is worthwhile to compare our results to work performed using biotin-modified DNA strands attached to a bilayer surface through a biotin−streptavidin coupling.19 The main difference between our two systems, apart from the chemical nature of the anchor and linker, is the placement of the anchor along the DNA sequence, resulting in duplex DNA that is parallel to the surface (the present study) or perpendicular to the surface (using the end-on biotinylated DNA strands). Large differences are seen in the QCM-D data upon hybridization. For our system, hybridization results in a lowering of the energy dissipation due to the stiffening of the DNA, although the change is small (15%). For end-on immobilized DNA, the stiffening upon hybridization increases the height of the DNA layer, resulting in an elongated and flexible structure, which induces a 30% increase in ΔD (a much softer film). The different anchoring strategies result in different diffusive properties in the membrane, which affects the hybridization rate. Therefore a kinetic comparison of hybridization for the two systems was not performed. To verify the binding behavior and hybridization of ss bi-pe upon addition of ss comp, complementary measurements to the QCM-D were performed with an optical surface sensitive technique, DPI. A sample of prehybridized ds bi-pe was measured for comparison (Table 2 and Supporting Information). DPI is an optical sensing technique from which the biomolecular mass of a layer can be obtained (ΔmDPI). This mass is smaller than the acoustically coupled mass measured by QCM-D (ΔmQCM), and the difference between the two masses can be used to estimate the water content of the surfaceassociated molecular film. Since the molecular weight of the

Figure 6. QCM-D frequency shifts for the melting and rehybridizatin of ds tri-pe bound to lipid membrane. From 80 to 145 min the system was heated to 80 °C followed by removal of dissociated strands by flowing buffer for 15 min, and then cooled to room temperature from 160 to 300 min. Melting of the double helix was verified by rehybridization with the complementary strand at 310 min, followed by rinsing with 10 mM SDS, water, and buffer.

the signal obtained after cooling is expected to correspond to a layer of ss tri-pe, which was verified by the subsequent addition of ss comp resulting in hybridization accompanied by frequency and dissipation shifts similar to those observed upon hybridization of ss tri-pe at 60 °C, yielding a layer of ds tri-pe. 1951


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the structure, and form a completely new pattern by addition of different DNA strands. Although binding was irreversible in buffered solution, it was possible to remove 85% of the bound construct by flowing water over the membrane. This demonstrates an exciting new approach to DNA nanotechnology where ionic strength can be utilized to modify binding and surface coverage.

Lowering of the ionic strength in a solution of DNA is known to lower the melting temperature due to an increased single-strand repulsion caused by dissociation of positive counterions, and has previously been demonstrated for DNA attached to a gold surface via a thiol modification.31 To investigate whether exchanging the bulk fluid from buffer to water (dramatically lowering the ionic strength) could melt the double helix of porphyrin−DNA on the membrane surface, water was flowed over a saturated layer of ds bi-pe (see Supporting Information). Upon changing the bulk fluid back to buffer, ΔD−Δf plots showed values consistent with a layer of ds bi-pe, at 15% coverage. There was no indication that strands of ss bi-pe were present on the surface. In effect, a removal of strands was observed, not melting as expected. Desorption of ds bi-pe is attributed to removal of associated ions resulting in larger repulsive forces between adjacent molecules of negatively charged double-stranded DNA. Increased DNA−DNA repulsion due to a lowering in ionic strength has previously been shown to affect the coverage of DNA attached to gold via a thiol linker.32 Here we show that the intermolecular repulsion expels DNA from the membrane surface, whereas the intramolecular repulsion (denaturing of DNA) is not observed.

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ASSOCIATED CONTENT

S Supporting Information *

QCM-D binding data, QCM-D hybridization at half coverage, FRAP diffusion measurements, DPI binding data, QCM-D data for removal of bound construct, and calculation of molecular area. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS This research was funded by The Swedish Research Council (VR) through the Linnaeus program SUPRA. The authors are grateful for stimulating discussions with Rickard Frost and Dr. Marcus Swann.

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CONCLUDING REMARKS Zinc−porphyrin anchored DNA with a hydrophobic linker has been shown to bind to supported lipid membranes consisting of DOPC. Binding of a single-stranded construct revealed that a soft layer consisting of ∼2 times as many anchored molecules was formed compared to binding of double-stranded constructs. The binding was irreversible and at least 1 order of magnitude stronger compared to liposomes. Such strong binding is likely to be necessary in future applications if the addressability of DNA is to be utilized using the sequential addition of ligands, or for the sequential construction of DNA nanopatterns at the membrane surface, where a flow over the membrane is necessary. We envisage using complex DNA structures to position redox active porphyrins with subnanometer precision. The DNA thereby acts as a pattern template for the porphyrin, which can irreversibly modify either surface attached molecules, or electron accepting groups on the lipids themselves, thereby transferring the pattern from the DNA to the surface. Current work is being conducted to reversibly restrict the movement of the porphyrin−DNA in the membrane using temperature-dependent phase transitions in specific lipid mixtures. To use DNA as a template, it must be oriented parallel to the membrane, which is accomplished using the novel anchoring strategy described in this work. We demonstrated that a double-stranded linear construct can be formed either by direct addition of the construct, or by addition of anchored single strand, followed by hybridization at the membrane surface with the complementary sequence. This hybridization can be followed using ΔD−Δf plots, clearly showing that the layer formed consists only of double-stranded constructs. This is the first demonstration of applying ΔD−Δf plots to verify that hybridization is complete. Melting of the DNA duplex at the membrane surface by heating to 80 °C was also demonstrated. Although melting of membrane anchored DNA has been shown previously, our method is unique in that the melted strands were removed from solution, while the anchored strand remained in the membrane. It was then possible to rehybridize the DNA at the membrane by addition of new complementary strands. Using the same anchor, it should therefore be possible to form a DNA nanopattern, melt

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REFERENCES

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Reversible Hybridization of DNA Anchored to a Lipid Membrane via

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