Paramagnetic Decoration of DNA Origami Nanostructures by Eu3+

Article pubs.acs.org/Langmuir

Paramagnetic Decoration of DNA Origami Nanostructures by Eu3+ Coordination Lars Opherden,†,‡ Jana Oertel,§ Astrid Barkleit,§ Karim Fahmy,*,§ and Adrian Keller*,†,⊥ †

Institute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf, P.O.B. 510119, 01314 Dresden, Germany ‡ Technische Universität Dresden, Mommsenstraße 13, 01069 Dresden, Germany § Institute of Resource Ecology, Helmholtz-Zentrum Dresden-Rossendorf, P.O.B. 510119, 01314 Dresden, Germany ABSTRACT: The folding of DNA into arbitrary two- and threedimensional shapes, called DNA origami, represents a powerful tool for the synthesis of functional nanostructures. Here, we present the first approach toward the paramagnetic functionalization of DNA origami nanostructures by utilizing postassembly coordination with Eu3+ ions. In contrast to the usual formation of toroidal dsDNA condensates in the presence of trivalent cations, planar as well as rod-like DNA origami maintain their shape and monomeric state even under high loading with the trivalent lanthanide. Europium coordination was demonstrated by the change in Eu3+ luminescence upon binding to the two DNA origami. Their natural circular dichroism in the Mg2+- and Eu3+-bound state was found to be very similar to that of genomic DNA, evidencing little influence of the DNA origami superstructure on the local chirality of the stacked base pairs. In contrast, the magnetic circular dichroism of the Mg2+-bound DNA origami deviates from that of genomic DNA. Furthermore, the lanthanide affects the magnetic properties of DNA in a superstructure-dependent fashion, indicative of the existence of superstructure-specific geometry of Eu3+ binding sites in the DNA origami that are not formed in genomic DNA. This simple approach lays the foundation for the generation of magneto-responsive DNA origami nanostructures. Such systems do not require covalent modifications and can be used for the magnetic manipulation of DNA nanostructures or for the paramagnetic alignment of molecules in NMR spectroscopy.

1. INTRODUCTION The DNA origami technique1 enables the fast, high-yield synthesis of arbitrarily shaped 2D and 3D nanostructures by exploiting the strong specificity of Watson−Crick base pairing. It employs a long, single-stranded DNA scaffold which is folded into the desired shape by a suitable (i.e., complementary) set of designed short synthetic oligonucleotides, called staple strands. The sequence of each staple strand is designed to facilitate multiple binding events with different segments of the scaffold strand, thus forcing the scaffold to fold into an arbitrary shape which is determined by the sequences of the individual staple strands. Since its introduction in 2006, the DNA origami technique has attracted enormous interest in various research fields.2 Considerable effort has been spent on the utilization of DNA origami for a number of applications in molecular sensing,3,4 biophysics,5,6 and drug delivery.7−9 DNA origami nanostructures have furthermore been used as “molecular breadboards” for the controlled arrangement of functional entities including plasmonic nanoparticles, 10,11 quantum dots,12,13 fluorophores,14,15 carbon nanotubes,16,17 and a large variety of proteins.18−20 The latter particularly enabled the study of chemical21,22 and biochemical reactions23−25 at the singlemolecule level. The intrinsic diamagnetic properties of DNA origami, on the other hand, have attracted attention because © 2014 American Chemical Society

they allow aligning biomolecules in solution, thereby facilitating, for instance, nuclear magnetic resonance (NMR) structure determination of large macromolecules through residual dipolar couplings.26,27 For the same purpose, the association of paramagnetic lanthanides such as europium with natural or engineered chelating sites on a biomolecule has been exploited as well.28 In this work, we have attempted to combine the synthesis of differently shaped DNA origami with a lanthanide-mediated “paramagnetic decoration” at the intrinsic phosphorus sites. Although straightforward, the approach is not trivial for DNA origami because the trivalent lanthanides induce DNA condensation and can interfere with DNA backbone structure. We have identified the optimal conditions for the magnetic functionalization of two representative DNA origami nanostructures by postassembly coordination of the DNA backbone with Eu3+ ions. Trivalent lanthanide ions such as Eu3+ and Tb3+ are known to exhibit distinct magnetic and luminescence properties29−31 which make them attractive for various applications ranging from fluorescent labeling29 and magnetic resonance imaging30 to the synthesis of single-molecule magnets.31 Here, we explore Received: March 24, 2014 Revised: June 15, 2014 Published: June 23, 2014 8152

dx.doi.org/10.1021/la501112a | Langmuir 2014, 30, 8152−8159

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Eu3+ coordination of DNA origami as a tool for synthesizing paramagnetic DNA nanostructures. DNA is known to be diamagnetic at room temperature,32 while trivalent europium is a classic Van Vleck paramagnet.33 Although Eu3+ is nonmagnetic in its ground state 7F0, the first excited magnetic state 7F1 becomes populated at sufficient thermal energies, which results in an effective magnetic moment of ∼3.4 μB at 300 K.33 Therefore, Eu3+ coordination can be used to efficiently alter the magnetic properties of DNA, as exemplified by the alignment of Eu3+-coordinated DNA molecules by external magnetic fields in NMR studies.34 Here, we explore the conditions under which a high load with Eu3+ ions can be achieved on DNA origami without affecting either their shape or their monomeric state. Natural polynucleotides exhibit well-known metal-binding properties,35 whereas little is known about metal-binding properties of DNA origami. Here, two representative DNA nanostructures, i.e., a planar triangular and an elongated tube-shaped DNA origami, were used to further address the potential role of superstructure for both the stability and magnetic properties of the resulting molecular assemblies. Maintaining stability and preventing aggregation in the presence of Eu3+ is not trivial, because the “native” Mg2+ is a key factor in preserving DNA origami structure. In fact, the divalent ion becomes replaced by the trivalent Eu3+. While Mg2+ engages predominantly in watermediated contacts to the DNA, lanthanides often exhibit inner sphere coordination.36,37 In addition, Eu3+ can more strongly reduce electrostatic repulsion between DNA origami, facilitating their aggregation. The attempted paramagnetic decoration of DNA origami thus requires the structural investigation of Eu3+−DNA interactions for which we have used complementary spectroscopic techniques and atomic force microscopy (AFM). The Eu3+ coordination was studied by luminescence spectroscopy, whereas AFM and circular dichroism (CD) spectroscopy revealed an intact nanostructure and DNA conformation. Using magnetic circular dichroism (MCD), we have identified magnetically induced chirality of electronic transitions that are unique to the DNA origami and were not seen in genomic DNA. These MCD bands are specifically affected by Eu3+ binding in a superstructure-dependent fashion reflecting different interdouble strand geometries of the lanthanide-binding sites. Such structural differences were further confirmed for the two DNA origami structures by determining the coordination number of the bound Eu3+ ions using time-resolved laser fluorescence spectroscopy (TRLFS). Our approach may provide an efficient tool for the magnetic manipulation of DNA origami nanostructures independently of covalent modifications. The degree of diamagnetic alignment increases linearly with the anisotropic susceptibility, while it increases with the square of the paramagnetic moment.38 Therefore, magnetic alignment of a “lanthanide-decorated” DNA origami can be superior over diamagnetically aligned DNA structures and may serve functions in nanostructure manipulation as well as in molecular alignment for spectroscopic structural investigations.

6.7, Sigma-Aldrich). To this end, the scaffold (10 nM) was mixed at a molar ratio of 1:30 with each of the staple strands in pure 10 mM MgCl2 solution in a total volume of 200 μL. The solution was then annealed by gradually decreasing the temperature from 80 to 4 °C in 90 min using an Eppendorf Mastercycler Personal. All staple strands were purchased from Metabion. Two different DNA origami structures have been chosen for studying Eu3+ coordination, i.e., a 2D triangular design and a 3D sixhelix bundle. The triangular DNA origami introduced by Rothemund1 is composed of three identical trapezoids with a planar arrangement of parallel double helices that meet under a 60° angle. This design has been selected because the triangles are more rigid than, for instance, the also common rectangular DNA origami and less prone to aggregation by intermolecular stacking interactions, as they do not exhibit any exposed bases at their edges. The triangular DNA origami can thus be considered as dispersed 2D plates with an edge length of ∼130 nm. The DNA origami six-helix bundle was adopted from Bui et al.12 It features six parallel helices of 412 nm length arranged in a tubelike shape and, due to its large persistence length, behaves like a rigid rod. Europium coordination was achieved by adding 200 μL of EuCl3 solution (50 or 100 μM, pH 5.2, Alfa Aesar) to the annealed, unpurified DNA origami sample followed by centrifugation for 10 min through an Amicon Ultra-0.5 mL filter (100 kDa MWCO, Millipore) at 6000 rpm. After addition of another 300 μL of EuCl3, the sample was filtered a second time with the same parameters and finally recovered from the filter. For Mg2+-coordinated reference samples, the samples were filtered with 10 mM MgCl2 solution following the same protocol. DNA origami concentrations after spin filtering were determined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific) and typically ranged from 40 to 100 nM, corresponding to 600−1500 μM in DNA-phosphate. We have also attempted DNA origami assembly both in EuCl3 solution and EuCl3-containing TAE buffer. However, no DNA origami could be observed by AFM after spin filtering. We believe that, due to its very high affinity to the phosphates in the DNA backbone, one Eu3+ ion may coordinate simultaneously with up to three phosphates in the same oligonucleotide, which causes structural distortions of the staple strands and thereby prevents assembly. AFM Imaging. For AFM imaging, 2 μL of the sample was mixed with 40 μL of 10 mM MgCl2 solution and adsorbed for 5 min onto a freshly cleaved mica surface. After washing the mica sample with 1 mL of Milli-Q water and drying it in a stream of nitrogen, it was imaged in air using a Bruker MultiMode 8 scanning probe microscope operated in tapping mode and Tap150Al-G probes (BudgetSensors). Steady State Luminescence Spectroscopy. Steady state luminescence spectroscopy was performed at 395 nm excitation (detector voltage 850 V) using a LS55 luminescence spectrometer (PerkinElmer). Emission spectra were recorded in the range from 550 to 650 nm at a speed of 100 nm/min using a 430 nm cutoff filter. To improve the signal-to-noise ratio, the spectra have been averaged over 16 individual measurements. In all luminescence spectra, an exponential scattering background has been subtracted to enable comparison of absolute peak intensities. Time-Resolved Laser Fluorescence Spectroscopy. The TRLFS measurements for Eu3+ were carried out with a pulsed flash lamp pumped Nd:YAG-OPO laser system from Continuum as described previously39 at an excitation wavelength of 394 nm and a constant time window of 1 ms for all measurements. Static and timeresolved luminescence spectra of Eu3+ were recorded in the range 565−650 nm (1200 lines mm−1 grating, 0.2 nm resolution, 2000 accumulations) and 440−780 nm (300 lines mm−1 grating, 0.7 nm resolution, 200 accumulations), respectively. For time-resolved measurements, 41 spectra were recorded with 40 μs separation. The lifetimes were calculated by fitting the integrated luminescence signal to a sum of exponential decay functions:

2. EXPERIMENTAL SECTION Assembly and Coordination of DNA Origami. The DNA origami nanostructures were assembled from the M13mp18 viral scaffold (New England Biolabs) with 7249 nucleotides as described previously.24 However, in order to avoid unwanted complexation of the Eu3+ ions with residues from the typically used TAE buffer, DNA origami assembly was performed in pure 10 mM MgCl2 solution (pH

E(t ) =

∑ Ei × e−t/ τi i

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(1)


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Here, E is the total luminescence intensity at time t, Ei the luminescence intensity of the species i at the time t = 0, and τi the corresponding lifetime. The number of water molecules in the first coordination shell was determined from the luminescence lifetimes τ (in ms) applying the linear relationship developed by Horrocks and Sudnick40 and the resultant empirical formula from Kimura et al.:41

n(H 2O) ± 0.5 = 1.07 × τ −1 − 0.62

(2)

CD and MCD Spectroscopy. A JASCO J-815 CD spectrometer equipped with a GMW 3470 electromagnet was used for CD and MCD spectroscopy. The spectra were recorded in the range from 185 to 320 nm at a speed of 200 nm/min and averaged over eight individual measurements. To account for differences in concentration, the CD spectra were normalized to the absorption of DNA at 260 nm that was measured in parallel. MCD spectra were obtained by subtracting CD spectra recorded with a magnetic field of 2.25 T applied parallel and antiparallel to the optical path. The magnetic flux of the magnet was calibrated using the MCD signal of 150 mM CoSO4 at 510 nm.42 To enable better comparison between the individual spectra, the MCD spectra were normalized to the most prominent difference band around 250 nm/270 nm and shifted vertically to yield zero intensity at 320 nm for the Mg2+ spectra. Thereby, spectral shapes can be compared directly and independently of variations in concentration or effective magnetic field strength. For reference measurements, genomic double-stranded (ds) DNA from salmon testes (Calbiochem) has been used.

Figure 1. DNA origami concentration measured by UV−vis spectroscopy as a function of EuCl3 concentration. The solid line represents a logistic regression fit to the data. The AFM images have a size of 1 × 1 μm 2 and were taken at the indicated EuCl 3 concentrations.

binding affinity to DNA than Mg2+, charge neutralization may be reached at even lower Eu3+ concentrations. In order to verify Eu3+ coordination of the DNA origami at low EuCl3 concentrations with δ < 0.4, luminescence spectroscopy was employed. The relative intensities of the main luminescence bands of Eu3+ at 590−593 and 614−616 nm corresponding to the magnetic and electrical dipole transitions 5D0 → 7F1 and 5D0 → 7F2, respectively, are known to depend strongly on the interaction with its surroundings.44,48 Figure 2a depicts the luminescence spectrum of the 100 μM EuCl3 solution (dotted line) in the spectral range from 560 to 640 nm. In agreement with previously reported measurements,48 the pure EuCl3 spectrum shows two luminescence bands with the band centered around 591 nm being more intense than the one around 615 nm, which is indicative of a highly symmetric water complex. In the presence of DNA origami, however, the symmetry-forbidden 615 nm band increases drastically in intensity (see Figure 2a, solid line). As expected, no luminescence is observed within the relevant wavelength range for DNA origami in pure 10 mM MgCl2 solution (Figure 2a, broken line). The increase in the intensity of the 5D0 → 7F2 transition results from the displacement of water molecules in the hydration shell of Eu3+ by inner-shell ligands43,44 which therefore evidence the efficient coordination of the lanthanide by the DNA origami. Furthermore, the Eu3+ coordination was found to be very stable, since the luminescence spectra did not change significantly upon addition of MgCl2 up to a final concentration of 10 mM (see Figure 2b). This agrees with the 600 times stronger binding of Eu3+ to nucleic acids compared to Mg2+ as reported in the literature.49 Although no influence of Eu3+ coordination on the DNA origami superstructure was detectable with AFM (see Figure 1), we have assessed possible structural effects on a molecular level by CD spectroscopy. Figure 3a shows the natural CD spectra of DNA origami in 10 mM MgCl2 (broken lines) and 100 μM EuCl3 (solid lines), respectively. Both spectra are of similar shape and intensity and resemble published CD spectra of dsDNA.50 For comparison, reference CD spectra of genomic dsDNA are shown in Figure 3b. In MgCl2 solution, very similar CD spectra were obtained for the triangular DNA origami and the genomic dsDNA with both the long wavelength maxima and the zero crossings having almost identical intensities and

3. RESULTS AND DISCUSSION Among the multiple DNA binding sites for trivalent lanthanide ions,36,37,43−45 the strong Lewis acid Eu3+ exhibits a high affinity to the oxygens of the DNA backbone phosphates.36,43−45 Eu3+ coordination may thus affect the DNA origami nanostructures by reducing electrostatic repulsion between neighboring helices similar to the frequently used Mg2+ ions which are competing with the Eu3+ ions for the same binding sites.46 Before binding site saturation is reached, however, DNA condensation is likely to occur, e.g., due to local charge inversion.45 In order to find optimal conditions for the structurepreserving paramagnetic decoration, 2D triangular DNA origami have been incubated with Eu3+ concentrations ranging from 50 μM to 1 mM. A DNA origami sample in 50 μM EuCl3 was split into identical aliquots and the Eu3+ concentration adjusted individually at identical volumes. After 30 min of equilibration, the DNA origami concentrations in the different samples were measured by UV−vis spectroscopy. As can be seen in Figure 1, the DNA origami concentration remains rather constant with increasing Eu3+ concentration until a sudden drop is observed at ∼200 μM EuCl3, which indicates the condensation of the DNA origami. From the individual concentrations, the onset of condensation was determined to occur at a ratio of δ ∼ 0.4 Eu3+ ions per phosphate group, similar to results from mass spectroscopy of Eu3+-bound oligonucleotides.47 Condensation occurs at the charge neutralization point, i.e., at 0.33 bound Eu3+ ions per phosphate, which is reached here at 200 μM EuCl3 for ∼500 μM total phosphate of the DNA origami (∼35 nM). About 80% of the Eu3+ ions in solution can thus bind to the DNA origami before condensation. Over the full range of Eu3+ concentrations studied here, intact DNA origami triangles can be observed by AFM (see Figure 1), thus demonstrating the stability of the Eu3+-bound DNA origami even in the absence of Mg2+ ions. Note that, if DNA origami assembly is performed with monovalent ions such as Na+ which have a much lower 8154


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DNA origami which deviate from the B form of the genomic dsDNA.51 In the presence of EuCl3, the CD spectra of both the DNA origami and the genomic dsDNA show a reduction of the negative intensity in the range from 230 to 260 nm as compared to the signature measured at 10 mM Mg2+. This can be attributed to the different ionic strengths of the buffer solutions.50 Structural alterations upon Eu3+ coordination such as unstacking and condensation on the other hand would result in an overall decrease of the CD signal52 and can thus be ruled out. The effect of Eu3+ coordination on the magnetic properties of the triangular DNA origami was assessed by MCD spectroscopy. DNA exhibits an intrinsic MCD with the three main bands E1u, B1u, and B2u at ∼210, ∼250, and ∼270 nm, respectively.53 In contrast to the rather similar natural CD, the MCD spectrum of the triangular DNA origami nanostructures in MgCl2 solution depicted in Figure 4a (broken line) differs

Figure 2. (a) Steady state luminescence spectra of pure 100 μM EuCl3 solution, triangular DNA origami in 100 μM EuCl3 (δ = 0.1), and triangular DNA origami in 10 mM MgCl2 (75 nM), respectively. (b) Steady state luminescence spectra of triangular DNA origami in 100 μM EuCl3 (δ = 0.14) before and after addition of MgCl2 to a final concentration of 10 mM. The vertical lines at 591 and 615 nm correspond to the Eu3+ dipole transitions 5D0 → 7F1 and 5D0 → 7F2, respectively.

Figure 4. MCD spectra of (a) triangular DNA origami, (b) DNA origami six-helix bundles, and (c) genomic dsDNA in 10 mM MgCl2 and 100 μM EuCl3 solution. The Eu/phosphate ratio was δ = 0.14 (a, c) and δ = 0.07 (b).

from that of genomic dsDNA in Figure 4c (broken line). Although the zero-crossing at 260 nm is conserved, the negative peak at 270 nm is blue-shifted as compared to genomic dsDNA. In addition, a weak but highly reproducible signal is observed between 300 and 315 nm. On the high frequency part of the MCD signature, the broad positive lobe of genomic dsDNA at 247 nm is replaced by two peaks at 253 and 237 nm. The MCD between 220 and 240 nm is generally more intense and more structured for the DNA origami triangles than for genomic dsDNA. These differences are thus specific for the base pair geometry in the DNA origami. In order to elucidate whether the altered MCD is related to the predominantly parallel

Figure 3. CD spectra of (a) triangular DNA origami nanostructures and (b) genomic dsDNA in 10 mM MgCl2 and 100 μM EuCl3 solution. For both DNA origami and genomic dsDNA, the Eu/ phosphate ratio was δ = 0.14.

positions, respectively. Only the negative lobe with a minimum at around 245 nm is slightly smaller for genomic DNA. Since both DNA samples possess almost identical GC contents of 41−43%, this spectral difference is likely to be caused by the novel structural motifs introduced by the staple strands in the 8155


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model of lanthanide-dependent mixing of excited states in DNA, a geometrical interpretation of the MCD data at the molecular level is at present not possible. Since the Eu3+decorated DNA origami stay intact, it is reasonable to correlate their spectral differences with their salient structural differences. Therefore, we propose that the involvement of the multiple nonplanar DNA strands in Eu3+ coordination in the six-helix bundles causes the persistence of magnetic interactions between double strands also in the presence of the lanthanide. The notion that different Eu3+ binding geometries lead to the different MCD signatures was further tested by TRLFS. The luminescence spectra of 100 μM EuCl3 with the two DNA origami show the characteristic features of Eu3+ luminescence upon complex formation, namely, the strong increase of the hypersensitive 5D0 → 7F2 transition at about 615 nm and the appearance of the symmetry forbidden 5D0 → 7F0 transition at around 578 nm (see Figure 5). The measurement of the

packing of dsDNA in the DNA origami or additionally dependent on its overall shape, the effect of Eu3+ coordination has also been investigated for a 3D six-helix bundle which differs distinctly from the planar geometry of the 2D triangular DNA origami. Figure 4b (broken line) shows the MCD of the six-helix bundle DNA origami complexed with Mg2+. In fact, the spectrum exhibits all the MCD features that have been observed with the triangular origami. In agreement with the dependence of CD and MCD on nearest neighbor interactions, the spectra indicate that it is indeed the general property of the close laterally packed double strands within the DNA origami which leads to the almost identical MCD signatures for the two structures. Upon coordination with Eu3+, deviations from the MCD spectra of the Mg2+-bound states are observed for genomic dsDNA as well as for the two DNA origami. The two peaks of the MCD spectrum of the triangles between 235 and 260 nm are replaced by a single maximum at 247 nm, evidencing that the coordination of Eu3+ affects the magnetic properties of the triangular DNA origami. The frequency of the negative peak at 270 nm coincides with that of genomic dsDNA, and also the unusual positive feature of the DNA origami at long wavelengths is abolished by the lanthanide. Thus, the MCD in the Eu3+-bound state of the triangular DNA origami is almost indistinguishable from that of the corresponding genomic dsDNA. The situation is different for the six-helix bundles, where the short-wavelength absorption is little affected by Eu3+, whereas the long-wavelength side becomes more structured (a negative peak at 267 nm and a new shoulder at 280 nm), indicative of an additional magnetically sensitive optical transition in the Eu3+-bound state. Besides these shapedependent interactions of the lanthanide with the two DNA origami, the abolishment of the magnetically induced chirality above 300 nm is a general feature of the Eu3+-bound DNA origami. In summary, the MCD in the absence of Eu3+ reveals a shape-independent magnetically induced chirality in the DNA origami in the 230−250 nm range and at wavelengths above 300 nm that is not seen in genomic dsDNA. We assign both spectral features to the massive parallel alignment of base pairs in the two DNA origami, as compared to the purely longitudinal alignment in the genomic dsDNA. The appearance of natural CD signals at long wavelengths (>300 nm) has been described for condensed DNA forms.54,55 Remarkably, however, no natural CD has been observed for the DNA origami here, in agreement with the absence of light-scattering aggregates. Instead, the signals above 300 nm, observed here, are exclusively induced by the magnetic field. The MCD discriminates the two DNA origami nanostructures only in the Eu3+-bound but not in the Mg2+-bound state. This suggests that the MCD originates in the magnetic mixing of excited states and that this mixing is altered upon binding of the paramagnetic lanthanide. In the case of the planar triangular structure, the MCD suggests a similar geometry as in Eu3+bound genomic dsDNA, indicating that Eu3+ largely abolishes the magnetic interaction between the closely packed double strands in the plane of the DNA origami. This further implies that the particular in-plane arrangement of the double helices in 2D DNA origami does not have a dominant effect on the MCD of the Eu3+-bound state. In the tube-like structure of the sixhelix bundle, however, the altered MCD signature most likely results from a different Eu3+ coordination. Due to the lack of crystallographic data of DNA origami and of a theoretical

Figure 5. Luminescence spectra of 100 μM Eu3+ complexed with triangular DNA origami (δ = 0.14, top) and DNA origami six-helix bundles (δ = 0.07, middle). For comparison, the spectrum of pure EuCl3 is shown as well (bottom).

luminescence lifetime shows for both DNA types a biexponential decay, indicating the coexistence of two different binding modes (Figure 6). For uncomplexed EuCl3, the luminescence lifetime is ∼110 μs corresponding to nine water molecules in the first spherical coordination shell.56,57 Upon complexation with the DNA origami, two prolonged luminescence lifetimes are observed. The first lifetime with τ1 = 137 ± 24 μs (triangles) and τ1 = 183 ± 13 μs (six-helix bundles) corresponds to 7.4 ± 1.4 and 5.3 ± 0.4 water molecules, respectively, in the first coordination sphere, indicating that the two DNA origami structures bind differently. The second lifetime with τ2 = 387 ± 25 μs and τ2 = 333 ± 44 μs is even longer, corresponding to less remaining water molecules around the Eu3+ ion (about 2.2 ± 0.2 H2O with sixhelix bundles and 2.7 ± 0.4 H2O with triangles). The different lifetimes represent different binding modes of Eu3+ and thus confirm the results from MCD spectroscopy. Additional outer8156


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is required at 300 K. With the reported magnetic moment33 of Eu3+ of 3.4 μB, this could theoretically be achieved with only three Eu3+ ions if they bind with maximal anisotropy (for example, two Eu3+ are sufficient to induce residual dipolar couplings in a DNA 15-mer at 17 T).34 Our study shows that Eu3+ binds to DNA origami in a shape-dependent manner. This supports the existence of binding site anisotropy which is required for magnetic alignment58 and particularly expected for partial inner sphere coordination44 demonstrated here by the TRLFS measurements and most convincingly by the appearance of the symmetry-forbidden 5D0 → 7F0 transition in both DNA origami. The maximum number of Eu3+ ions that can bind to DNA before charge neutralization causes condensation is one-third of the number of nucleotides. In the case of the DNA origami, this amounts to more than 4000 Eu3+ ions. Therefore, even a small relative anisotropy among the Eu3+ binding sites can suffice to generate a Δμ that exceeds that in typical NMR experiments on residual dipolar couplings of magnetically aligned molecules. Importantly, the shape conservation of the Eu3+-decorated DNA origami shown in the AFM images demonstrates that the DNA origami is a stable scaffold that resists the large restructuring processes that are usually induced by trivalent cations in dsDNA.59 Thus, Eu3+decorated DNA origami may in general exhibit a high potential for magnetic manipulation and may be further optimized in their shape for this purpose.

Figure 6. Luminescence lifetimes of Eu3+ complexed with triangular DNA origami (δ = 0.14, circles) and DNA origami six-helix bundles (δ = 0.07, squares). The solid lines are fits to the data according to eq 1 with the resulting lifetimes τi given in the graph.

sphere complexes are not ruled out but cannot be distinguished from free aquoions. On the basis of the NMR structure34 of DNA-bound Eu3+ and in line with previous Eu3+ luminescence data for polynucleotides,36,37 we assign the prolonged luminescence lifetimes of DNA origami-bound Eu3+ to specific binding sites. Although the spectroscopic differences between the two DNA origami probably arise from contributions from several of such sites in each sample, we rule out that they are caused by the average signal of a diffuse cation cloud. The basis for the stable binding is the inner shell coordination of the lanthanide. Since neither TRLFS nor MCD provide atomic resolution, the origin of the different Eu3+ binding modes observed for the different superstructures remains speculative. One obvious structural difference that could influence Eu3+ coordination is the nonplanar arrangement of double helices in the six-helix bundle which might enable coordination of a single Eu3+ ion inside the tubular shape to three neighboring double helices. In addition, the out-of-plane arrangement of the helices may also be accompanied by a different degree of twist strain, causing more or less structural distortions and thus different deviations from the natural B form in the two DNA origami structures. Finally, the different distances between neighboring crossovers in the triangular and the tubular DNA origami could further result in interhelix gaps of different sizes which again might affect Eu3+ binding. In order to establish a clear superstructure-binding relationship, the crystal structures of the different Eu3+−DNA origami complexes need to be determined. Nevertheless, the significantly different lifetimes τ1 detected for the different DNA origami nanostructures in our TRLFS measurements are unambiguous proof of different Eu3+ binding modes. We have estimated the potential of the Eu3+-decorated DNA origami for their paramagnetic alignment. The latter is described by the order parameter S = ⟨0.5(3 cos2 θ − 1)⟩ with the brackets denoting the average over all orientations of the magnetic moment relative to the magnetic field. Using Boltzmann statistics, S = (ΔμB/kT)2/15, where Δμ = (μ∥ − μ⊥) is the anisotropy of the paramagnetic moment.38 S values on the order of 10−6−10−5 have been achieved at B fields of 9− 10 T with the purely diamagnetic alignment of small aromatic compounds leading to residual dipolar couplings in their NMR spectra.38 To reach similar alignments by paramagnetic decoration already at 1 T, a Δμ of 10 Bohr magnetons (μB)

4. CONCLUSIONS In summary, we have demonstrated the formation of Eu3+decorated DNA origami nanostructures that preserve their structure in the absence of Mg2+. Magnetically induced chirality in two differently shaped Mg2+-bound DNA origami was found to be independent of the superstructure and thus predominantly determined by the general properties of DNA origami, i.e., the dense lateral packing of double helices. In the Eu3+bound state, however, the MCD is altered, as expected for the perturbation of local magnetic fields by paramagnetic decoration. Remarkably, the MCD is superstructure-dependent which implies different geometries of the Eu3+ binding sites residing between the differently packed double strands in the planar DNA origami triangles versus the tube-shaped six-helix bundles. Such an anisotropy of the Eu3+ binding sites is a key prerequisite for paramagnetic alignment, as seen, for instance, with Eu3+-decorated DNA aptamers.34 Similarly, and dependent on the supramolecular shape and interstrand geometry, Eu3+decorated DNA origami may promote the magnetic alignment in nanodevices or confer alignment to associated molecules in NMR studies.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Address ⊥

Chemical Engineering and Macromolecular Chemistry, University of Paderborn, Warburger Str. 100, 33098 Paderborn, Germany.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 8157


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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank S. Tsushima for theoretical support, M. Kuczera for technical assistance, J. Lindner for discussions, and I. Bald for helpful comments and a careful reading of the manuscript. A.K. acknowledges financial support from the Alexander von Humboldt foundation.

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