Electrotransfection of Polyamine Folded DNA ... - ACS Publications

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Electrotransfection of Polyamine Folded DNA Origami Structures Aradhana Chopra, Swati Krishnan, and Friedrich C. Simmel* Physik-Department E14, Technische Universität München, 85748 Garching, Germany S Supporting Information *

ABSTRACT: DNA origami structures are artificial molecular nanostructures in which DNA double helices are forced into a closely packed configuration by a multitude of DNA strand crossovers. We show that three different types of origami structures (a flat sheet, a hollow tube, and a compact origami block) can be formed in magnesium-free buffer solutions containing low ( 0.5 M27) and even in the water-free eutectic solvent glycholine (a 4:1 mixture of glycerol and choline chloride).28

ecent advances in DNA nanotechnology have demonstrated that DNA, in addition to its paramount biological role, can be successfully utilized as a programmable building material for the self-assembly of biomolecular nanostructures1 and machinelike dynamical nanodevices.2 DNA nanostructures have been designed and assembled to realize essentially any geometry using methods such as DNA origami3,4 or DNA brick assembly.5 The successful origami technique in particular is based on the hybridization of a large number of short DNA “staple” strands, typically of ∼42 nucleotides (nt) in length, with a long single-stranded DNA molecule termed the “scaffold”, which is commonly derived from the single-stranded genome of phage M13mp18 and has a length between 7 and 8 kilobases (kb).6 Upon hybridization of staples and scaffold, distant parts of the scaffold strand are connected, “folding” it into a particular spatial arrangement. A completely folded, layered3,4 (nonwireframe) DNA origami structure consists of a large number of DNA double helices packed together in parallel at a close distance and connected via multiple strand crossovers. The overall structure of the origami complexes can be freely chosen and is determined by the routing of the scaffold and, thus, the choice of the staple sequences. DNA nanostructures have been used as platforms for the precise spatial organization of functional molecules such as fluorophores,7,8 aptamers,9 antibodies,10,11 membrane receptors,12 lipids,13,14 and inorganic nanomaterials15,16 through oligonucleotide coupling, which has led to the emergence of a wide range of applications for DNA nanotechnology. One of the potential disadvantages of DNA as a molecular building material, however, is its polyanionic nature; with a linear charge density of ∼1 e/0.17 nm (or 5.9 e/nm), double-stranded DNA (dsDNA) is one of the most highly charged polymers © XXXX American Chemical Society

Received: August 25, 2016 Revised: September 7, 2016

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DOI: 10.1021/acs.nanolett.6b03586 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. Electrotransfection of Spd nanostructures in mammalian cells.

Figure 2. Folding of different DNA origami nanostructures in a buffer containing the condensing agent spermidine (Spd3+). (A) Schematic illustration of folding process using different buffers. (B) Agarose gel electrophoresis demonstrating the folding of DNA nanostructures using different concentrations of Spd3+ required for folding 50 nM DNA nanostructures. L, 2 log ladder; S, scaffold; C, DNA nanostructures folded in MgCl2 (control). Spermidine concentrations in the numbered lanes were: lane 1, 0 μM; 2, 50 μM; 3, 100 μM; 4, 200 μM; 5, 300 μM; 6, 400 μM; 7, 500 μM; 8, 600 μM; 9, 700 μM; 10, 800 μM; 11, 900 μM; 12, 1 mM; 13, 5 mM; and 14, 10 mM. (C−E) TEM micrographs of DNA nanostructures of different shapes and lattice types folded in the condensing agent, Spd3+. Inset: class-averaged single-particle image. Scale bar: 100 nm.

tion agent spermidine (Spd3+). In biology, DNA condensation is essential for the packaging and storing of biological DNA within the small confines of the cell nucleus, sperm heads, the bacterial cytoplasm, or in viral capsids.34−36 DNA condensation in vivo is facilitated by histone proteins, protamines, and polyamines. Biogenic polyamines such as putrescine, spermidine, and spermine are linear multivalent compounds that are fully protonated under physiological pH and thus electrostatically interact with DNA and RNA, and they have been extensively used to study the DNA condensation process in vitro.20 One of the most striking features is the formation of tightly packed toroidal DNA complexes upon condensation,21 and it has been found that the condensation transition is strongly influenced by the presence of other monovalent or divalent cations.37,38 Polyamines have also been shown to enhance the stability of DNA duplexes with respect to heat denaturation.39 On the basis of these properties, polyamines have also been used as a component of nonviral gene delivery systems, either as simple cationic oligomers or in conjugation

The requirement for high salt (specifically, magnesium) concentrations poses a particular challenge for potential applications of DNA nanostructures in cell biology and nanomedicine. In a recent systematic investigation of the sensitivity of DNA origami nanostructures to in vitro cell culture media,23 it was found that the denaturation of the structures due to depletion of divalent cations was a major problem, and they also suffered from degradation by nucleases. These effects could be remedied, e.g., by inhibiting the nucleases and supplementing the media with magnesium. Other approaches to protect and stabilize DNA nanostructures under physiological conditions involve the utilization of polyethylene glycol29 as a crowding agent, decoration with viral coat proteins,30 or complexation with transfection agents such as lipofectamine, which also facilitate cellular uptake of the structures.30−33 In the present work, we investigate an alternative approach for the folding of DNA origami structures under low-ionicstrength conditions that utilizes the trivalent DNA condensaB


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Figure 3. Purification of Atto647N-labeled Spd nanostructures (42HB variant) folded in Spd3+ using size-exclusion chromatography. (A) Schematic representation of SEC method using a series of two columns packed with different volumes of sephacryl resins. Unpurified Atto647-labeled Spd nanostructures are loaded in column 1, and eluate 1 collected after centrifugation is fed as a sample to column 2. The eluate from column 2 contains purified Atto647-labeled Spd nanostructures. (B) Agarose gel electrophoresis to check the quality and yield of purified products. Overlay of the typhoon scanner image and SyBr gold stained image to identify the dye-labeled DNA from unlabeled DNA. L, 2 log ladder; S, scaffold; 1, unpurified Spd nanostructures; 2, eluate 1; and 3, eluate 2. The recovery yield was found to be ≈85−90% (eluate 2, band in well 3) (calculated from band intensities measured using Fiji).

tional folding buffer with typical millimolar concentrations of MgCl2 (C). For reactions with Spd3+ concentrations up to 700 μM (well 9, Figure 2 B), bands corresponding to unstructured or partially structured DNA complexes moving with the scaffold (S) were observed. Faster migrating species emerged at higher concentrations of condensing agent ([Spd3+] ≈ 800− 1000 μM), which corresponded to the correctly folded origami structures (red box in Figure 2 B). As shown in Figure S2, origami tubes and rectangles were also successfully folded in Spd3+-containing buffers. In addition to gel electrophoresis, the correct assembly of the Spd nanostructures was also verified using negative- and positive-stain TEM (Figure 2C−E) (cf. the Transmission Electron Microscopy section in the Supporting Information). Circular dichroism (CD) measurements of 42HB folded in traditional buffer and Spd3+ resulted in the same CD signatures, indicating that no major conformational distortion was induced by the presence of Spd3+ (Figure S3). We found that previously established purification protocols working with a high-salt background were not suitable for our Spd-folded structures, and we therefore developed a dedicated purification procedure based on size-exclusion chromatography (SEC)44 (Figure 3; cf. the Modification and Purification of DNA Origami Structures section and Table S1 in the Supporting Information). As shown in Figure S4, the concentration of Spd3+ required for folding was approximately linearly proportional to the concentration of the DNA origami structures. This observation was consistently made for all the three DNA origami nanostructures used in this work. In contrast, Mg nanostructures folded at the same magnesium concentration irrespective of the DNA concentration (Figure S4). The 42 helix bundles are formed by ∼7500 bp using a 3-fold excess of staple strands over scaffold during the assembly. For a scaffold concentration of 50 nM, this corresponds to a concentration of DNA phosphates of ∼1.5 mM in the folding solution. In this case, the optimum Spd3+ concentration for folding was found to be ∼800 μM (i.e., cspd/cDNA,phos ≈ 0.5), slightly overcompensating the charge of the phosphates (see also the discussion below). At higher trivalent salt concentrations, a whitish precipitate was observed during folding, suggesting aggregation and potentially condensation of the DNA (Figure S5C). Aggregates were also visible in the agarose

with lipids. Compaction of DNA with polyamines enhances its cellular uptake and thus improves transfection efficiency.40 In this study, we demonstrate the “biocompatible’’ folding of DNA origami structures at approximately physiological spermidine concentrations ([Spd3+] < 1 mM). For comparison, the concentration of polyamines in mammalian cells ranges from ≈0.1−3 mM, with typical spermidine concentrations on the order of 2 mM, which includes both bound and free species. The fraction of each may vary considerably depending on the cell state and type,41 and concentrations of f ree spermidine have been estimated to lie in the range between 15−200 μM.42,43 In contrast, magnesium concentrations ([Mg2+] ≈ 10−20 mM) in typical origami folding buffers are much higher than those prevailing in cells.23 An important consequence of the utilization of comparatively low Spd3+ concentrations is the considerably reduced ionic strength and conductivity of the folding buffer. We also found enhanced stability of Spd3+assembled DNA origami structures when subjected to high electric fields in comparison to that of DNA nanostructures assembled in traditional magnesium-based buffers. Origamistabilizing Spd3+ buffers were found compatible with the buffer conditions required for electroporation experiments, and we exploited this feature for intracellular delivery of DNA origami structures into mammalian cells via electroporation (Figure 1). Assembly of DNA Origami Structures in Spermidine. We used the trivalent condensing agent spermidine to facilitate the assembly of three types of DNA origami nanostructures with different shapes and helix packing densities: a twistcorrected regular Rothemund rectangle,3 a hollow DNA origami tube, and a 42 helix bundle (42HB)27 in a one-pot reaction. Detailed information about structures, folding, cadnano designs, and DNA staple sequences of the different structures are provided in Figure S1 and Tables S1−S3. A schematic representation of the folding process and the chemical structure of the condensing agent is given in Figure 2 A). To assess the yield and success of the origami folding process, reaction products were analyzed using agarose gel electrophoresis (AGE) (cf. the Agarose Gel Electrophoresis section in the Supporting Information). For the gel shown in Figure 2 B, 42 helix bundles (50 nM) were assembled in varying Spd3+ concentrations ranging from 0 to 10 mM. As a positive control, nanostructures were also formed in convenC


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Figure 4. Effect of electric fields on the stability of DNA nanostructures folded in different buffers. (A,B,E,F) Schematic representation and representative TEM micrographs exhibiting the fate of DNA nanostructures (42HB) folded in MgCl2 and Spd3+, respectively, before and after application of electric field. (C,G) Tables with details of different electric fields applied, associated parameters, and fate of the nanostructures. (D,H) Agarose gel electrophoresis showing the fate of Mg and Spd nanostructures (42HB variant) to different electric fields in comparison to controls. L, 2 log ladder; S, scaffold; 1, control (no electric field); 2, E = 250 V/cm; and 3, E = 500 V/cm. Scale bar: 100 nm.

DNA concentration. At low DNA concentrations, larger amounts of polyamines were required when monovalent salt were added to the solution. The experimental situation is different in the case of Spd3+supported DNA origami folding. Here, we start with the 7.5 kb long single-stranded scaffold strands and a large number of shorter staple strands in Spd3+-folding buffer without added salt at roughly millimolar (i.e., “intermediate”37) DNA monomer concentrations and subject them to a thermal annealing ramp. It is conceivable that in this case, Spd3+ initially supports annealing of the staples with the scaffold and thereby becomes incorporated inside of the closely packed origami structures in a similar manner as that assumed for DNA condensates. Spd3+ will not only interact with the origami structures, however, but also with the excessive staple strands remaining free in solution. In the context of our experiments, we therefore cannot precisely determine the ratio between Spd3+ and the phosphates in the origami structures, but it is roughly on the same order. Most strikingly, the Spd3+ concentration required for folding displays the same linear trend with DNA concentration as in the condensation experiments. Aggregation and precipitation of the DNA structures at higher Spd3+ concentrations (Figure S5) indicates that the optimum Spd3+ concentration for origami folding might be just below or right at the condensation transition. It has been shown in high-resolution TEM and AFM studies3,50,51 that in DNA origami structures, the DNA helices only come close at the crossover points, whereas they otherwise avoid each other due to electrostatic repulsions. The estimated distance between neighboring DNA helices in origami structures is in the range of ∼2.5−3 nm,3,50,51 which is actually very similar to the spacing in DNA condensates, for which the

gels (blue box in Figure 2 B, wells 13−14) and TEM micrographs. It has been demonstrated before that multilayered DNA origami nanostructures can be folded in buffers containing high concentrations of monovalent salt (i.e., Na+), which are roughly 100 times larger than the magnesium concentrations contained in typical folding buffers.27 In contrast, the optimum Spd3+ concentrations found here are about 10 times smaller than these. It is well-known that multivalent cations (with charge ≥ + 3e), such as the polyamines spermidine and spermine, interact differently with DNA than with cations with smaller charge.17,20,22 In particular, whereas Na+ and Mg2+ cannot induce DNA condensation in aqueous solution, multivalent ions mediate like-charge attractions between DNA duplexes and can thus condense DNA into tightly packed structures. Various models have been put forward to explain the peculiar interactions between polyamines and double-stranded DNA molecules, which assume a correlated arrangement of the counterions along the charged DNA rods and also bridging between helices by the rodlike polyamines.22,45 Experimental evidence from CD, IR, and NMR studies46,47 as well as MD simulations48 suggest that Spd3+ preferentially interacts with the phosphates46 but also with the bases in the major and minor groove.47 Phenomenologically, we find similarities between the DNA condensation process and Spd3+ origami folding. In classical experiments studying DNA condensation by polyamines,37,49 150 bp long double-stranded DNA molecules were mixed (with or without additional monovalent salt) with the condensing agent, and the concentration range was determined, in which DNA precipitation was observed. The polyamine concentration inducing precipitation was found to increase smoothly with the D


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Figure 5. Electroporation of Atto647N-labeled Spd nanostructures (42HB) in Jurkat cells. (A) Schematic representation illustrating the distribution of Spd nanostructures at different subcellular z sections and typical images obtained by confocal microscopy. The cell volume was imaged as z sections each of width 0.5 μm, accounting for the entire volume of the cell (diameter ∼15 μm). (B) Confocal micrographs of Jurkat cells showing the overlay of fluorescence channels (green, blue, and red) and bright field (BF) taken at different z sections within the cell. The Spd nanostructures were present at different z positions inside the cell. The cell organelles lysosome (green) and cell membrane (blue) were stained to confirm the localization of Spd nanostructures in the cytosol. Blue blobs (shown by white arrows) correspond to the cell membrane, which appears at different z positions for different cells due to variable cell size (∼11−15 μm) of the cells and differential attachment of cells to the chamber. Red blobs represent the Spd nanostructures that did not enter the cell via electroporation and are either stuck to the membrane or to the chamber. This may be due to the interaction of the Spd nanostructures with the collagen coating of the chamber that facilitates the adhesion of cells.59 The red blobs disappear with increasing z, i.e., more into the interior of the cell, thus supporting this interpretation. Scale bar: 10 μm.

assessed using agarose gel electrophoresis, which clearly showed destruction of Mg-folded structures (Figure S10) while indicating that Spd-folded structures remained intact (Figure S11). According to Figure S9, Mg-folded 42HB structures sustained the long (τ = 12 ms) electric fields pulses only below E ≈ 125 V/cm, and the short-duration (τ = 0.4 ms) pulses below E ≈ 4000 V/cm. During these pulses, approximately the same amount of charge flows through the chamber, which is proportional to the product of the maximum field strength and the time constant, Emax · τ (note that, roughly, Q = ∫ I dt = ∫ j A dt = ∫ κ E A dt = κ Emax A ∫ exp(−t/τ) dt = κ A Emax τ, where j is the current density, κ is the conductivity, and A is the electrode area). In Figure 4, we focus on electric field pulsing conditions suitable for electroporation of DNA into mammalian cells (e.g., Jurkat), i.e., using 0.4 cm electroporation cuvettes, relatively low voltages, and longer pulse durations (Figure 4). As evidenced by the TEM micrographs in Figure 4B,F, Mg nanostructures indeed disintegrate in the electric fields, while Spd nanostructures appear unaffected. The tables in Figure 4C,G summarize the impact of the pulsed electric fields on the stability of Mg2+and Spd3+-folded 42HB, and the corresponding agarose gels are shown Figure 4D,H. A similarly increased stability of Spd nanostructures compared to that of Mg nanostructures was also found for the other origami structures investigated (data not shown). We also investigated electroporation parameters typically used for bacterial transfection, in which higher field strengths using smaller 0.1 cm cuvettes and shorter pulse lengths are applied. For the applications of strong electric field pulses, higher-resistance samples are preferred to avoid large electrical currents or electrical breakthrough in the sample chamber, which could damage cells and instrumentation. We therefore diluted Mg- or Spd-folded DNA origami structures (42HB) in a commonly used high-resistance medium (10% v/v glycerol in water) and again subjected them to a range of electric field

DNA duplexes were found packed on a hexagonal lattice with a Bragg spacing of ∼3 nm.52,53 Our observation that the overall size of the Spd nanostructures is not changed with respect to the Mg-folded structures thus cannot distinguish between conventional origami folding or internal “condensation” (Figures S6−S8). Stability of Spermidine-Folded DNA Helix Bundles in Electric Field Pulses. We next explored the possibility to introduce DNA nanostructures into live cells by electroporation. We therefore subjected 42HB origami structures folded in Mg2+ and Spd3+ buffers to the high electric field pulses generated by an electroporation apparatus. The electroporation instrument used is based on the capacitor discharge method.54 In this method, a capacitor C (which can be selected to be 36 μF in high-voltage (HV) mode and 1050 μF in low-voltage (LV) mode) is first charged to a set voltage and then discharged via a resistor Ri, which is connected in parallel to the sample cell (load resistance RL). The voltage across the capacitor and, thus, the electric field in the sample cell therefore exponentially decays over time with a characteristic RC time τ, where the effective resistance R is given by R = RiRL/(Ri + RL). In preparation of electrotransfection experiments with mammalian cells (see below), we used the standard cell culture medium RPMI as the sample medium, which has a relatively high conductivity of ∼1.5 S/m.55 This translates to a sample chamber load resistance of RL ≈ 12 Ω and, thus, time constants of τ = 0.4 ms in HV and τ = 12 ms in LV mode. To investigate the influence of electric pulses, we diluted Mg- or Spd-folded DNA origami structures (42HB) in RPMI (cf. the DNA Nanostructure in Electric Field section in the Supporting Information). We then subjected the sample to a range of electric field strengths in both LV and HV modes. As summarized in the comprehensive table given in Figure S9, Spd nanostructures were found to sustain the exposure to different electric fields under all applied conditions, while Mg nanostructures disintegrated in higher fields and for longer pulse durations. The range of stability of the structures was E


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and employing lipid-based transfection agents.30−32 Our results demonstrate that with origami nanostructures folded in and stabilized by the polyamine spermidine, electroporation could be an interesting alternative approach toward intracellular delivery of such nanostructures, potentially providing access to other subcellular compartments than those typically achieved with the other methods. Conclusions. We have demonstrated an approach for the folding and stabilization of DNA origami nanostructures in folding buffers containing the naturally occurring condensing agent spermidine. Compared to folding in standard Mg-based buffers, folding takes place at much-lower ionic strengths with a spermidine concentration at least 10-fold lower than typical magnesium concentrations. Our results suggest an optimum concentration of Spd3+ roughly compensating the phosphate charges in the origami structures. At much-higher concentrations, aggregation and precipitation of the origami structures set in. We also studied the robustness of Spd-folded origami structures with respect to electric field pulses as they are typically applied in electrotransfection experiments. We found Spd nanostructures to be stable under all conditions investigated, whereas Mg nanostructures disintegrated at all electric field strengths and pulse durations relevant for cellular delivery via electroporation. We also demonstrated electrotransfection of Spd-folded origami structures into Jurkat cells. Spermidine folding is an interesting alternative to high-ionicstrength folding buffers whenever the presence of a large concentrations of mono- or divalent cations is detrimental in the context of the application in mind. Moreover, spermidine appears to stabilize DNA origami structures in low-conductivity buffers as well as in electrical fields, which could be of general interest for electric field manipulation of DNA origami objects. Spd-folded origami structures appear to be stable in cell lysate, which is important for the various nanomedical applications that have been proposed for origami structures and potentially also for the creation of nucleic acid nanostructures in vivo. Notably, the natural intracellular concentration of polyamines is on the same order as that used in our origami folding experiments.

strengths, both in LV and in HV mode (Figure S12). In the low-conductivity case, R ≈ Ri, and thus, the time constants are increased to τ ≈ 0.16 s and τ ≈ 5 ms, respectively. As before, the Spd-folded structures sustained all pulse conditions, while Mg-nanostructures disintegrated at high field strengths and for longer pulse durations. In the presence of an electric field, counterions are expected to flow around and also through the DNA origami structures. This effect has been previously studied by molecular dynamics simulations in the context of DNA-based membrane channels56 and also for DNA origami plates attached to solid-state nanopores57 in a 50 mM MgCl2/1 M KCl electrolyte solution. It is thus likely that for Mg-folded origami structures, the application of a strong electrical field pulse strips away the stabilizing Mg2+ counterions, resulting in the disintegration of the structures due to internal electrostatic repulsion; in the electroporation buffer, the stripped counterions cannot be replaced by a sufficient amount of other divalent ions. In contrast, Spd3+ ions are more tightly attached to the DNA origami nanostructures and stabilize the structures, even in the presence of a strong electric pulse. This is consistent with the view that the elongated polycation Spd3+ stabilizes the origami structures by making inter- and intrastrand polyelectrolyte bridges.22 Electrotransfection of Spd Nanostructures in Jurkat Cells and Confocal Microscopy. Utilizing the stability of Spd-folded origami structures under electroporation conditions, we next electrotransfected Jurkat cells with 42 helix bundles fluorescently labeled with the dye Atto647N. Confocal micrographs of live Jurkat cells electroporated with Atto647N-42HB taken at different z positions are shown in Figure 5. After electroporation, the nanostructures were found to be present in various subcellular volumes. Intracellular localization of the nanostructures was confirmed by cellular organelle staining, in which lysosomes were stained in green and the cell membrane in blue. Judged from the confocal images, Spd nanostructures were found to be predominantly localized in the cytosol. Additional data (confocal micrographs and z-section videos of cells) showing cellular uptake of Spd nanostructures are provided in Figure S13 and Videos 1 and 2, respectively. In contrast, no internalization of Mg origami structures via electroporation was observed, suggesting that the nanostructures were destroyed when the electric field was applied (Figure S14). In control experiments, we were unable to detect Atto647N fluorescence when only dye-labeled strands were transfected (Figure S15). We generally found a good cell viability after application of the electroporation procedure (cf. the Cell Viability Assays section and Figure S16 in the Supporting Information). In cases where cells did not survive due to irreversible rupture of the cell membrane, we found incorporation of a larger amount of DNA nanostructures, potentially by binding to the ruptured membrane. On the basis of the confocal microscopy images, we could not unequivocally address the question of intracellular integrity of the Spd nanostructures. We therefore also assessed the stability of the origami structures in cell lysate as previously described in ref 58. The nanostructures were in fact found to be stable in a large range of cell lysates of different densities (cell number/mL; cf. the Stability in Cell Lysate section and Figure S17 in the Supporting Information) Previous transfection experiments with DNA nanostructures have been performed via receptor-mediated endocytosis using nanostructures decorated with CpG sequences or viral proteins

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ASSOCIATED CONTENT

* Supporting Information S

Additional information on experimental methods, agarose electrophoretic gel images, TEM micrographs, confocal microscopy images and DNA sequences are given. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b03586. Additional details on experimental methods. Figures showing cadnano diagrams of the three DNA origami nanostructures used in this work; class-averaged TEM images of DNA origami nanostructures folded in Spd3+ and agarose gel electrophoresis for screening of Spd3+ concentrations required for folding 50 nM DNA origami nanostructures; circular dichroism spectra of 42HB folded in traditional buffer containing magnesium and in Spd3+; relation between the concentration of folding buffer and concentration of DNA origami nanostructures to be folded; folding reactions and corresponding TEM micrographs for 42HB at different concentrations of Spd3+; negative-stain TEM micrographs of 42HB, tube, and tCRRO folded in traditional buffer containing F


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magnesium (A) and Spd3+; effect of different electric field strengths on the stability of 42HB folded in different buffers; agarose gel electrophoresis showing the stability of Mg nanostructures (42HB) diluted in RPMI and subjected to a range of electric field strengths both in LV and HV mode; agarose gel electrophoresis showing the stability of Spd nanostructures (42HB) diluted in RPMI and subjected to a range of electric field strengths both in LV and HV mode; effect of different electric field strengths on the stability of Mg and Spd nanostructures diluted in high-resistance sample buffers (10% v/v glycerol in water) and using smaller path length cuvettes (0.1 cm); confocal micrographs of Jurkat cells with Atto647N-labelled Spd nanostructures (42HB) internalized via electrotransfection; results from an electroporation experiment with Mg nanostructures and Jurkat cells and with Atto 647N labelled DNA strands in Jurkat cells; cell viability assays; and stability of Spd nanostructures (42HB) in Jurkat lysate. Tables showing sequences of DNA staples for different DNA origami nanostructures generated by cadnano software. (PDF) Video assembled from 30 confocal microscopy z-sections taken from Jurkat cells in 0.5 μm steps (corresponding to Figure 5B). (AVI) Video assembled from 30 confocal microscopy z-sections taken from Jurkat cells in 0.5 μm steps (corresponding to Figure S13). (AVI)

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Corresponding Author

*Phone: +49 (0)89 289 11611; fax: +49 (0)89 289 11612; email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Marie Curie Initial Training Network EscoDNA (European School of DNA Nanotechnology, GA no. 317110) and the Deutsche Forschungsgemeinschaft (SFB 863 TP A8). The authors thank Florian Praetorius for different variants of M13mp18 scaffold and Jonathan List for designing the origami tube.

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