Controlling and Monitoring Orientation of DNA Nanoconstructs on

Its extraordinary self-assembly property, with potential to form nonperiodic structures with unique addressability, makes DNA ideal for fabrication of...
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Controlling and Monitoring Orientation of DNA Nanoconstructs on Lipid Surfaces Erik P. Lundberg, Bobo Feng, Amir Saeid Mohammadi, L. Marcus Wilhelmsson, and Bengt Nordén* Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden S Supporting Information *

ABSTRACT: Its extraordinary self-assembly property, with potential to form nonperiodic structures with unique addressability, makes DNA ideal for fabrication of advanced nanostructures. We here demonstrate the controllable tethering of a hexagonal DNA nanostructure in two distinct orientations at the lipid bilayer of a liposome functioning as a soft-matter support. With polarized light (linear dichroism) applied to the flow-aligned liposomes, we show that the construct is preferentially in a parallel alignment with the lipid surface when two anchors are attached while with one anchor only a perpendicular orientation is observed.

1. INTRODUCTION

Attaching DNA networks to surfaces may also provide an important step toward bridging the gap between a molecular bottom-up approach and the lithographic top-down strategy of nanotechnology, for example, deposition of DNA on mica surfaces by divalent cations such as Mg2+ and visualization with atomic force microscopy.1 More controlled integration between prefabricated DNA nanoconstructs and lithographically patterned surfaces is possible by lithographic surface-patterning techniques with specific binding pockets for DNA nanostructures,52,53 manifesting higher orders of organization and predictability compared to more disordered structures on unmodified solid supports. Yet another example from the literature demonstrated the possibility to bridge spatially separated Au discs using thiol-modified DNA nanotubes54 representing direct integration between bottom-up and topdown approaches to nanotechnology. DNA structures have also been applied as masks in shadow nanolithography, resulting in nanometer thin etchings in Si(100) wafers or as a lithographic mask.55,56 These examples illustrate that DNA nanotechnology may become an integral part of molecular lithography enabling means for high-precision etching.

The inherent rigidity and effective recognition power of Watson−Crick base pairing of DNA enable a programmable self-assembly of supramolecular structures with unprecedented precision. On this basis, DNA nanotechnology has developed rapidly during the past two decades: as crystal-like periodic arrays,1−4 addressable networks,5−9 three-dimensional solitaires,10−15 mechanical devices,16−21 computational logics,22−28 and the DNA origami approach.29−37 Two-dimensional (2D) DNA networks may be used as templates in chip-based devices for controlling processes on the nanometer scale between molecular functionalities. The level of control depends on the precision of the DNA-based template. Fully addressable networks are a prerequisite for freedom in localization of active units with well-defined spatial separation. Examples of such systems are photonic-based circuits38−42 and assembly lines for chemical synthesis.43−46 Fully addressable DNA nanonetworks have been assembled either using unique building blocks with exclusive recognition patterns5,7,9 or utilizing inherent addressability upon folding of genomic m13 in the DNA origami approach.47−51 In addition to Watson− Crick base-pair hybridization, triple-helix recognition sites may also be used as addresses in prefabricated DNA duplex networks.8 © 2012 American Chemical Society

Received: January 18, 2012 Revised: November 26, 2012 Published: December 3, 2012 285

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Figure 1. Hexagonal (Hex) and linear (20-mer) DNA nanostructures tethered to a lipid bilayer via one or two anchors of glyceryl-bis-C16hexaethyleneglycol (C16-HEG) (shown at bottom) covalently attached to the 3′ and 5′ ends of 10 bases long recognition oligonucleotides.

should be integrated with the lipid bilayers, these should be parallel with the surface, thus probably requiring several anchoring points. Hexagonal nanostructures have been developed in our laboratory,68−71 for example, built from three-way branched oligonucleotides.8,9 A DNA nanostructure anchored to a lipid membrane via porphyrin moieties72−74 was suggested to need three anchors to keep it flat with the surface;75 however, severe spectral overlap with porphyrin absorption obviated DNA structure and orientation to be diagnosed.72,73 Avoiding the synthetic and functional complexity of porphyrins, optically transparent anchors are called for in general applications. We here study two hexagonal DNA nanostructures based on the hexagonal unit cell of Tumpane et al.,68 with each edge merely 10 bases long (3.4 nm). The nanoconstructs are intended to display orthogonal orientations when tethered to a lipid bilayer: one with a single lipid anchor (HexA1) and one with two lipid anchors on opposite sides of the hexagonal core (HexA2) that will give, respectively, perpendicular and parallel orientations of the construct relative to the membrane surface (Figure 1). Using polarized light (linear dichroism) on flow-aligned lipid vesicles, we can verify the alignment of the nanostructure depending on anchorage.

While the primary focus so far has been on integration between DNA nanostructures and solid surfaces, for example, mica, Si, or Au, soft surfaces such as lipid bilayer membranes are less explored. The fluidity of surfaces formed through selfassembled amphiphilic lipids allow tethered units to freely diffuse in the 2D bilayer, maintaining a dynamic system. Studies in our group have previously shown that slightly positive lipid vesicles catalyze strand exchange between DNA duplexes.57,58 The hydrophobic interior of the membrane has also interesting chemical and physical properties that may be exploited for tuning purposes or as insulation between functionalities fixed to the two leaflets. Controllable integration between DNA nanostructures and lipid bilayers could therefore pave way for applications of dynamic systems with chemically heterogeneous functional units. Studies on DNA−lipid membrane conjugates, DNA tethered to lipid bilayers using cholesterol or lipid as anchors,59−64 have dealt with intermembrane connections mediated by DNA duplex hybridization vesicle−vesicle assemblies62,64 or vesicle−lipid bilayer (SLB).65,66 Anchored DNA has also been utilized to mimic the function of SNARE proteins promoting lipid vesicle fusion.59,67 Common to previous studies has been that tethered DNA duplex molecules are in a perpendicular orientation with respect to the membrane surface. If more complex DNA nanostructures 286

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Figure 2. UV melting characteristics for the hexagonal DNA nanostructure, free in solution without sucrose as well as tethered to a lipid vesicle, with either one (blue) or two (red) anchors, respectively. Insets are the first derivative of the melting curve. Melting temperatures (Tm) according to the peak value of the derivative are 40 and 30 °C for the DNA nanoconstruct when free in solution and bound to liposomes, respectively, for both HexA1 and HexA2. viscosity of the buffer is increased improving the orientation of the liposomes. An additional effect is substantially reduced light scattering due to matching of refractive indices between sucrose buffer and the lipid vesicle membrane. Any chromophore associated with the membrane in a specific orientation will subsequently display a corresponding LD signal. LD is defined as follows:

2. MATERIALS AND METHODS 2.1. DNA Constructs. The hexagonal DNA constructs are formed by hybridization of six oligonucleotides, each 10 bases long and mutually orthogonal with respect to sequence (see Figure 1 for details). Each stretch of 10 complementary bases is flanked by two unpaired thymines functioning as hinges in the final construct. A protruding stretch of 10 bases, with a spacer of two thymines, is complementary to a modified oligonucleotide that anchors the DNA nanostructure to the membrane surface. In this study, we make use of an amphiphilic motif, glyceryl-bis-C16hexaethyleneglycol (C16-HEG), covalently attached at the 5′ or 3′ end of oligonucleotides.76 The hydrophobic glyceryl-bis-C16 unit associates with the interior of the membrane, and the hydrophilic hexaethyleneglycol (HEG) is a spacer between the surface and the DNA molecule. 2.2. Membrane. The membrane in this study is a bilayer of zwitterionic double-tailed phosphocholine lipids (DOPC) assembled into unilamellar vesicles of approximately 100 nm in diameter (see the Supporting Information for details). The extensive size difference between the vesicle and the DNA nanoconstruct means that the surface curvature is negligible at the length scale of the construct, that is, the vesicle membrane may be regarded as approximately flat. The lipid vesicles and DNA nanostructures are assembled separately and mixed to form DNA−vesicle conjugates with an anchor/lipid ratio of 1:200. Given the size of a vesicle and the effective area of the phosphocholine headgroup,77 this molar ratio corresponds to an average occupancy of 400 DNA nanostructures per vesicle for constructs tethered with one anchor and 200 nanoconstructs in the case of two anchors. As earlier mentioned, DNA constructs with multiple anchoring points could potentially link separate vesicles together.59,62,67 The anchor/lipid ratio was chosen to avoid cross-linking, since we have previously shown that aggregation may be avoided by either decreasing the anchor/lipid ratio or the concentration of DNA−liposome conjugates.75 2.3. Linear Dichroism. Linear dichroism (LD) is a method to study molecular orientation. It is based on the preferential absorption of polarized light oriented in the same direction as the electric transition dipole moment of a given chromophore. Structural information may be obtained from LD about how the molecule is oriented with respect to the polarization of the incident light. Lipid vesicles are possible to orient in shear flow due to ellipsoidal deformation caused by shear forces from a rotating Couette cell.78,79 The addition of sucrose (50% w/w) as suggested by the earlier report78 plays an important role in this aspect. First and foremost, the

LD = A − A⊥

(1)

where A∥ and A⊥ are the absorption of parallel and perpendicular polarized light, respectively, in relation to a macroscopic axis in the laboratory, generally chosen as the preferred sample orientation axis. Getting quantitative data for the angular alignment and thus structural information about an oriented system, one usually makes LD independent of concentration and path length. The reduced linear dichroism (LDr), being normalized with respect to the isotropic absorption, LDr = LD/Aiso, relates the direction of the transition moment to the orientation axis. In this system, where DNA is oriented with respect to the surface of a macroscopically aligned lipid vesicle, three angles define the orientation of the system. First, the angle between the helical axis and the membrane normal is denoted α (Figure S4 in the Supporting Information). Second, the angle between the helical axis and the base stack, that is, the transition dipole moment of the macromolecule, is denoted β. The third angle, δ, is the angle between the membrane normal and the long axis of the deformed liposome, the latter assumed parallel to the macroscopic orientation axis. These angles determine LDr through the following factors: ⎛ 3cos2 α − 1 ⎞⎛ 3cos2 β − 1 ⎞⎛ 3cos2 δ − 1 ⎞ LD = 3S⎜ ⎟⎜ ⎟⎜ ⎟ 2 2 2 A iso ⎝ ⎠⎝ ⎠ ⎠⎝ (2) where Aiso is the isotropic absorption and S is an orientation factor. The orientation factor describes the degree of alignment of the lipid vesicles, deformed by the shear forces in the flow. S is 1 for perfect orientation of an infinitely elongated vesicle (= tube) and 0 for the isotropic case.78,79 The third factor in the equation is equal to −1/2, if we assume δ = 90° (tube) and that any deviation of membrane plane from this geometry be condensed into the orientation factor S. The second factor is also −1/2, to a good approximation, as the effective angle β between the helix axis and the various in-plane transition moments in nucleobases (averaged by their cos2 values) is 86° in the helical stack of B-DNA,80,81 hence a reasonable assumption. These angles yield the following expression for the LDr of DNA tethered to an oriented lipid membrane: LDr =

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Figure 3. LD spectra of DNA nanoconstructs tethered with lipid anchors to lipid bilayer vesicles (images of DNA simplified for clarity; true conformations consistent with LD data are presented in Figure 4). (left) Hexagonal DNA nanostructures, tethered with one (HexA1, blue) or two anchors (HexA2, red). (right) Linear 20-mer duplex tethered in two arrangements, either with one (20-merA1, blue) or with two anchors (20merA2, red).

Figure 4. Schematic of the DNA nanostructure in arrangements suggested by the LD results. The hexagonal unit cell is viewed as totally isotropic in both cases, and in contrast, the double-stranded 10-mer stretch of the anchors are anistropic, yielding an LD signal. The LDr values at 260 nm of HexA1 (blue) and HexA2 (red) are 0.00668 and −0.00469, respectively. The corresponding angles, in respect to the membrane normal, are 49.4° and 58.7°.

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3 S(3 < cos2 α > − 1) 8

bilayer under the present conditions. The UV absorbance traces of two sequential cycles thus reveals total reversibility for both HexA1 and HexA2 (data in the Supporting Information). In the case of HexA2, there is yet another conclusion to be drawn from this result, namely, the absence of undesired aggregation due to DNA-mediated intervesicle linking that would have given rise to a substantial increase in the optical density (OD) at low temperatures.62,75 The two opposite lipid anchors of HexA2 could potentially be an agent causing vesicle aggregation; however, the reversible melting profiles demonstrate that this does not occur, at least in the concentration regime of the present system. 3.2. Calibration of Orientation Factor. The orientation factor, S, may be calibrated using a probe for membrane orientation. To this end, retinoic acid was utilized, which has been shown to insert itself perpendicular to the membrane surface, aligned with the lipids in the bilayer, thereby representing the orientation of the membrane normal.82 With an LDr for retinoic acid of −0.010, the orientation factor of the system can be determined to be 0.066 (data in the Supporting Information). This can be compared with an orientation factor of 0.033 in the previously published report.82 The lower temperature (Couette cell maintained at T = 5 °C), yielding increased viscosity, is a likely source of the increased orientation. Note that S just represents the degree of order of the membrane; the degree of order of the construct is included in the ensemble average . 3.3. Orientation of DNA Nanoconstruct. The LD spectra of the DNA nanostructures tethered to lipid vesicles are displayed in Figure 3 together with the corresponding expected qualitative orientations. A clear difference can be seen between the LD spectra for the construct tethered with one anchor (HexA1, blue) and with two anchors (HexA2, red). As expected, when the hexagonal DNA construct is anchored at a single point (HexA1), it protrudes from the membrane, positioning the planes of the nucleobases preferentially parallel with the orientation axis, which results in a positive LD signal at 260 nm. HexA2 displays a mirrored spectrum, and the bases are consequently instead preferentially more perpendicular with respect to the orientation axis, thus indicating that the construct itself is rather aligned parallel with the membrane surface. These are the qualitative conclusions; we shall return with a quantitative discussion below. As a control, in order to check for possible optical artifacts, LD was also measured on samples having only a short linear double-stranded DNA attached by an anchor to the membrane. A 20-mer oligonucleotide duplex was tethered in two arrangements: either with one anchor (20-merA1, blue) or with two anchors (20-merA2, red), thus analogous to HexA1 and HexA2, respectively (Figure 3). The second anchor was replaced by a 10 bases long sequence without an anchor moiety, in 20-merA1, making the samples identical with respect to the number of bases. As seen in Figure 3, with only one anchor (20-merA1, blue) a positive signal is obtained while surprisingly the arrangement with two anchors (20-merA2, red) does not give rise to any significant LD. Qualitatively, the former displays the same orientation as its nanostructure equivalent, with a predominantly protruding orientation of the DNA relative to the membrane surface. However, the latter oligonucleotide with two anchors shows no detectable orientation. This could be explained by a tendency to bridge different liposomes making them form aggregates, thus preventing a defined DNA orientation. Aggregation of the

(3)

In this equation, by replacing cos2 α with , we have indicated that we deal with an ensemble average, the interpretation of which we shall return to below. The two extreme cases where the DNA molecule is either protruding from or perfectly aligned with the membrane surface, 0° and 90°, respectively, give rise to the following theoretical LDr: α = 0°:

α = 90°:

3 LDr = + S 4

3 LDr = − S 8

Notable is the asymmetric relation in signal strength, where the positive signal of the protruding arrangement gives rise to twice the value of the opposite arrangement. For arrangements between these two extremes, the angle can be determined using the measured LDr: ⎛ 8 LDr 1⎞ α = arccos⎜ + ⎟ 3⎠ ⎝ 9 S

(4)

For a DNA hexagon lying parallel with the plane of the lipid bilayer, obviously α = 90° for helical pieces of DNA, and LDr should be equal to −(3/8)S, or −0.38 when S = 1. The same is true if the horizontal DNA stems belonging to two anchors are included. By contrast, for a perpendicularly protruding single stem, we expect a positive LDr = +(3/4)S, or +0.75 if S = 1. In the case a hexagon is protruding, as shown to the right in Figure 4, perpendicularly to the lipid surface including one stem with anchor, three duplex turns of DNA will have α = 0° while four turns in the hexagon will have α = 60°. In total, when averaged appropriately over all bases, according to eq 3, this orientation of construct is therefore predicted to give LDr = +(15/56) S, or +0.27 for S = 1.

3. RESULTS AND DISCUSSION 3.1. Structural Integrity of the DNA Construct. A way of verifying the structural integrity of the hexagonal DNA nanostructure is to investigate its melting behavior (Tm). Thermodynamic characteristics of the hexagonal unit cell have previously been studied in our laboratory.68,70,71 There is a distinct pattern in the melting trace (absorbance at 260 nm versus temperature) of the DNA nanohexagon associated with the opening of the ring-closed structure manifested as a sharp peak in the first derivative of the melting function, which can be considered a footprint confirming the structural integrity of the system. HexA1 and HexA2 both display this feature of the melting trace, both when the DNA nanoconstructs are free in solution as well as when tethered to lipid vesicles (Figure 2). The Tm is approximately 40 °C for both constructs free in solution, which is slightly higher than a simple hexagon without tethers of very similar nucleotide sequence in Sandin et al.70 This is however not surprising given the higher ionic strength of the buffer ([Na+] = 500 mM compared to 200 mM in the previous report). When tethered to liposomes, this structural footprint is still present; however, a drop in Tm of about 10 °C is apparent for both HexA1 and HexA2. This destabilizing effect has been deduced to originate from the addition of sucrose (50% w/w) in the samples containing liposomes (details in Figure S2 of the Supporting Information). Results from gel electrophoresis also show that the DNA nanostructure is intact and ring-closed in the presence of sucrose (data in the Supporting Information). Even though the hexagonal nanostructure is generally significantly destabilized in the vesicle-tethered samples, the melting profiles confirm an overall maintained structural integrity upon anchoring to a lipid 289

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Figure 4 is a structural depiction of the anchored DNA nanostructures based on the angles for HexA1 and HexA2. The structural height of the two arrangements above the membrane surface may be estimated by a simple trigonometric calculation from the average dimensions of the hexagonal core and the linkers (approximately 10.5 and 3.5 nm for HexA1 and HexA2, respectively). The total projected particle size according to these estimations are marked with horizontal bars in Figure 5.

liposomes is confirmed by a specific peak in optical density in the UV melting curve of the sample and increased scattering (see Figure S3 of the Supporting Information). The DNA nanostructure exhibits flexibility, especially in the case of a single anchoring point. In order to further understand how the hexagonal structures may orient and subsequently give rise to LD, a sample with only one anchor and its complementary sequence (strands A1 and 3a in Figure 1) were tethered to a liposome. This short double-stranded stretch of 10 bases exhibits the same LD spectrum as HexA1 (see Figure S5 of the Supporting Information), implicating that the hexagonal DNA unit does not itself contribute significantly to LD but behaves rather isotropically when anchored at a single point to the lipid membrane. In part, this effect may be due to the small intrinsic LD of the hexagon (see estimate following after eq 4); in part, it is due to the anticipation that the geometry of the hexagon is distorted from planarity and furthermore flexibly linked to the stem oligonucleotide duplex. It is noteworthy that the DNA core unit seems to remain isotropic in HexA2, even though the opposite anchoring points could potentially give the construct a better alignment. The LD spectrum of HexA2 displays the same signal strength as for HexA1, albeit with opposite sign, thus implicating that it is the double-stranded stretch of 10 bases at each anchoring point that gives rise to the main LD. 3.4. Structural Information from LD Data. Knowing the degree of orientation in the system, it is possible to estimate the angles of the DNA nanoconstruct tethered in different arrangements from LDr values at 260 nm. Since only the double-stranded stretch of 10 bases in the anchor sequence and not all DNA bases orient and give rise to the preferential absorption of polarized light, the LDr values must be scaled with the number of anisotropic bases in the different DNA assemblies to give the angles of the parts that actually becomes oriented. Correcting the LDr of the three DNA samples tethered with only one anchor (HexA1, 20-merA1, and A1 + 3a) with this factor (20/154, 20/20, and 20/44, respectively) results in an apparent angle α of 49° for all three samples (data in the Supporting Information), recalling though that this angle only represents an ensemble average and thus a wide distribution of angles may be the case. If the DNA nanostructure tethered with two anchors to the lipid membrane, HexA2, is treated in the same manner, that is, with an isotropic core and two oriented double-stranded stretches on each side, the corrected LDr value corresponds to an angle of approximately 59°. In a case of perfect alignment with the membrane surface, the angle would approach 90°. The structural freedom of the isotropic core is not interpreted as total rotational flexibility but rather as wobbling of a semiplanar structure that yields no net orientation of the bases. One may ask whether the DNA constructs are quantitatively bound to the liposomes. If a considerable fraction were free in solution, the calculated LDr would have to be corrected for the smaller amount that is bound and is giving rise to LD. An effect of such a correction would be more extreme angles: better inplane orientation for the two-anchor construct and more perpendicular orientation for the one-anchor construct. However, titration studies (see the Supporting Information) show that, for both cases, the LD amplitude increases linearly with concentration. It would be unlikely that both systems would remain unsaturated recalling that two anchors are expected to increase affinity four times.

Figure 5. Hydrodynamic diameter measured by DLS for the DNA− liposome conjugates compared to bare liposome (squares). The anchor/lipid ratio is 1:200 for both HexA2 and HexA1. Horizontal bars mark expected diameter values for the hypothetical structures deduced from angles given by linear dichroism.

3.5. Confirmation by Dynamic Light Scattering. Using dynamic light scattering (DLS) to determine the hydrodynamic diameter of the liposomes shows a distinct variation in good agreement with the different DNA orientations obtained from LD data (Figure 5). An increased hydrodynamic diameter compared to bare liposomes is observed with HexA2, which is close to what would be expected for a parallel orientation of the hexagonal DNA core. Furthermore, the size of a liposome with HexA1 is found to be substantially larger than with HexA2, and the increased size is in agreement with the DNA nanostructure protruding from the lipid membrane. The structural flexibility associated with anchoring a single end of the construct, allowing it to wobble, could explain why the increase in hydrodynamic diameter is less than expected if the structure were rigid and pointing perfectly perpendicularly from the surface. Furthermore, the DLS results clearly show that aggregation due to vesicle cross-linking is not an issue for HexA2. All three samples exhibit low levels of polydispersity (PDI < 0.1) while formation of aggregates would have broadened the size distribution.

4. CONCLUSIONS In conclusion, we have demonstrated that complex DNA nanostructures can be tethered and aligned to a lipid membrane using two lipid anchors. The hexagonal DNA nanostructure maintains its structural integrity and exhibits a high level of flexibility even when linked to a lipid bilayer. Both DLS and LD data suggest that the DNA nanostructure is aligned with the hexagon parallel with the lipid membrane, when tethered with two opposing anchors. However, structural information from the LDr value proposes a slightly protruding orientation even when the nanostructure is tethered with two anchors. The 290

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flexibility of both anchoring units and the DNA nanostructure allows the DNA to deviate significantly from perfect alignment. In the future, one can imagine using more anchoring points in order to decrease flexibility of the DNA nanoconstructs keeping them on average closer to the membrane surface. We envision that the development of soft-surface DNA nanotechnology will pave the way for applications relying on highly dynamic systems, for example, structural scaffolds for artificial photosynthesis.

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ASSOCIATED CONTENT

S Supporting Information *

DNA sequences, experimental details, gel electrophoresis result, additional UV melting data, and more details concerning linear dichroism on this system, with determination of orientation factor and additional results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 (0) 31 772 3041; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Nils Carlsson is gratefully acknowledged for discussions regarding linear dichroism equations. This research is funded by the European Research Council (ERC Advanced Senior Grant to B.N.).

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