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Single Particle Tracking and Super-Resolution Imaging of MembraneAssisted Stop-and-Go Diffusion and Lattice Assembly of DNA Origami Susanne Kempter, Alena Khmelinskaia, Maximilian T. Strauss, Petra Schwille, Ralf Jungmann, Tim Liedl, and Wooli Bae ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04631 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Single Particle Tracking and Super-Resolution Imaging of MembraneAssisted Stop-and-Go Diffusion and Lattice Assembly of DNA Origami Susanne Kempter,1 Alena Khmelinskaia,2 Maximilian T. Strauss,1,

2

Petra Schwille,2 Ralf

Jungmann,1, 2 Tim Liedl,1 and Wooli Bae1†* 1. Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität, München, Germany. 2. Max Planck Institute of Biochemistry, Martinsried, Germany †Current address: Department of Bioengineering, Imperial College London, London, SW7 2AZ,

United Kingdom *Corresponding author. E-mail: [email protected] Abstract DNA nanostructures offer the possibility to mimic functional biological membrane components due to their nanometer-precise shape configurability and versatile biochemical functionality. Here we show that the diffusional behavior of DNA nanostructures and their assembly into higher order membrane-bound lattices can be controlled in a stop-and-go manner and that the process can be monitored with super-resolution imaging. The DNA structures are transiently immobilized on glass-supported lipid bilayers by changing the mono- and divalent cation concentrations of the surrounding buffer. Using DNA-PAINT super-resolution microscopy, we confirm the fixation of DNA origami structures with different shapes. On mica-supported lipid bilayers, in contrast, we observe residual movement. By increasing the concentration of NaCl and depleting MgCl2, a large fraction of DNA structures restarts to diffuse freely on both substrates. After addition of a set of oligonucleotides that enables three Y-shaped monomers to assemble into a three-legged shape (triskelion), the triskelia can be stopped and super-resolved. Exchanging buffer and adding another set of oligonucleotides triggers the triskelia to diffuse and assemble into hexagonal 2D lattices. This stop-and-go imaging technique provides a way to control and observe the diffusional behavior of DNA nanostructures on lipid membranes that could also lead to control of membrane-associated cargos. Keywords: DN nanotechnology, DNA origami, lipid membrane, diffusion, single-particle

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tracking, super-resolution microscopy

Recent advances in DNA nanotechnology allow the production of DNA structures with nanometer-precise structural control and with diverse biochemical functionalizations.1-5 For example, DNA nanostructures with the shape and functionality of membrane nanopores have been interfaced with lipid membranes by hydrophobic moieties that were conjugated at pivotal anchor points.6-13 With the help of hydrophobicity-mediated interactions between DNA structures and membranes, control of membrane curvature14-17 and bi-layer-supported structural assembly has been demonstrated, mimicking the natural functions of membraneassociated proteins.8-10 In other studies, electrostatic interactions between the negatively charged DNA backbone and polar or charged lipid head groups was suffice to promote membrane adherence.7, 9, 18 Irrespective of the mode of interaction, DNA nanostructuring has the potential to diversify the functions of artificial membranes and to control the shape and behavior of vesicles or cells by design. Due to inherent fluidity of membranes and the mobility of associated molecules, optical imaging of such dynamic systems faces severe hurdles. Reported diffusion constants of DNA nanostructures that are anchored to lipid bilayers lie in the range between 0.2 µm2/s and 4 µm2/s.7,

10, 15, 19

The structures are thus slow enough to be traceable in a conventional

fluorescence microscope.10, 19 However, diffraction-limited microscopes can only handle very sparse densities of labelled structures while membrane components often work in a cooperative manner in a densely packed environment, where structural organization is of major importance. At the same time the diffusing objects are orders of magnitudes too fast to be imaged with super-resolution techniques that would allow us to access structural details.20-24 Recent developments in stochastic super-resolution microscopy yielded resolutions high enough to probe the configuration of DNA-nanostructures in a large field of view. Nonetheless, as imaging times range between minutes and hours, super-resolution microscopy usually requires irreversible sample fixation. This results in the dilemma that diffusion-related mobility and behavior, which are important features of membrane-associated processes,25 are hard to follow with optical methods with high spatial resolution. High-speed AFM, an alternative non-optical method, allows visualization of nanoscale objects on lipid membranes8,

9

but bears some

disadvantages. As a contact-based method, the AFM tip may directly interfere with the object to probe. Additionally, this technique generally lacks the possibility of specifically labeling the


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objects of interest with different molecular probes for distinction. Here, we show that the concentration of ions in the surrounding buffer influences the interactions between lipids, glass and DNA, and thus can be used to reversibly control the diffusional behavior of the DNA structures. This is conceptually similar to controlling binding of DNA structures onto mica surfaces by changing the concentration of ions.26 We demonstrate, that by changing the concentration of ions in the sample, we can either optically follow the diffusion of DNA nanostructures on supported lipid bilayers (SLBs) or resolve structural details of their higher order structures with DNA-PAINT27 super-resolution imaging.

Results As a model system, we here used our previously published three-legged (triskelion) origami design, that was inspired by the protein clathrin.28 This structure is assembled from three identical DNA origami objects (monomers) and the resulting triskelia can further assemble into a hexagonal lattice (Figure 1, Supporting Information, Figure S1 and Table S1). The monomer is a 36 helix-bundle structure with the approximate shape of the letter Y with a truncated upper left arm.10 The remaining upper arm consists of 18 helices that are bent by 60°.3 With a set of trimerization oligonucleotides, this arm can be connected to the stump of the truncated arm of another monomer. When three monomers are connected, they form a triskelion structure with 3-fold rotational symmetry (Figure 1a, upper scheme). In the presence of a set of multimerization oligonucleotides, the triskelia further assemble into hexagonal lattices with 130 nm edges (Figure 1a, lower scheme). We confirmed the folding and assembly of both monomers and triskelia by agarose gel electrophoresis (Supporting Information, Figure S2) and transmission electron microscopy (Figure 1b).

We first analyzed the dependency of the diffusion behavior of the DNA nanostructures for different salt concentrations to find favorable conditions for DNA-PAINT imaging (Figure 2). For membrane attachment, three staple oligonucleotides were extended from the structure with an 18 nt overhang that hybridizes with cholesterol-modified oligonucleotides that were grafted on the SLBs. We labeled structures with eight Atto 647N dyes to determine their diffusion in a custom-built total internal reflection fluorescence (TIRF) microscope by single particle


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tracking (Figure 2a).29 In our DNA nanostructure storage buffer with 1TE supplemented with 5 mM MgCl2 and 150 mM NaCl, we found two distinct populations in terms of their diffusional behavior. 90% of the structures were diffusing with ~1 μm2/s (Figure 2b) which is similar to previously reported values on SLBs10, 19 and also resembles the diffusion speeds of membrane proteins.30 The second population exhibited a diffusion coefficient of ~10-4 μm2/s, which corresponds to fluctuations of 102 nm2/s. As our tracking precision is on the order of 10 nm, this diffusion value is our limit of detection and therefore indicates arrest of the structures (Supporting Information, Figure S3). It has been shown that divalent ions mediate the interaction between DNA and zwitterionic lipid molecules while Na+ ions can compete with Mg2+ ions to displace them.18 We therefore tested lower NaCl and higher MgCl2 concentration to increase the interaction among DNA nanostructures and lipid molecules, which should slow down the overall diffusion. Notably, most of the objects (97%) stopped their diffusion when the concentration of NaCl was decreased from 150 mM to 0 mM while the MgCl2 concentration was increased to 20 mM. The monomers started to diffuse again when we returned to 150 mM NaCl and 5 mM MgCl2 (Supporting Information, Figure S4). To explore this behavior further, we measured the diffusion of a single-layered DNA origami sheet (Fig. 2c, Supporting Information, Figure S5, and Table S2). Interestingly, most of these structures already stopped diffusing in a buffer with 0 mM NaCl and 5 mM MgCl2 (Fig. 2c). To test if the substrate that supported the SLBs affected the diffusional behavior of the DNA structures, we measured their diffusion coefficients also on mica-supported SLBs (Figure 2d, e). Different to the observation on SLBs on glass, the diffusion was unrestricted for almost all objects in the presence of 150 mM NaCl and 5 mM MgCl2. The observed diffusion constants were similar to those of freely moving objects on glass-supported SLBs (~1 μm2/s). Structures on mica-supported SLBs only slowed down when NaCl was removed from the buffer. Our further attempts to transiently stop the diffusion of DNA nanostructures on mica-supported SLBs failed even with 50 mM MgCl2 (Supporting information, Figure S6). Notably, the average diffusion exponent of traces recorded from structures on mica has a value of 1.02 ± 0.12 for all conditions, while the picture was more complex for bilayers on glass surfaces (Supporting information, Figure S7). Many traces on glass exhibit sub-diffusional behavior. We often observed particles stopping or wiggling around certain spots for short times and then returning to free diffusion. The diffusion exponents for such traces are well below 1 while the average of the freely moving structures is 1.01 ± 0.11 (Supporting information,


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Figure S7). We attribute this behavior to sub-micrometer defects in the membrane at these points and/or to increased surface roughness.

To confirm that DNA nanostructures are indeed immobilized even at a nanometer scale, we imaged immobilized monomer structures with DNA-PAINT (Figure 3a). For DNA-PAINT imaging, we designed three positions for a docking sequence to extend from the top of the origami monomer and removed Atto 647N labeling on the structure to reduce background fluorescence. After attaching the structures to a glass-supported SLB, we exchanged the buffer for ‘imaging buffer’ (20 mM MgCl2 and 0 mM NaCl) and added 20 nM of Atto 655 modified imager strands. These imager strands bind transiently to the 9 nt-long docking sequences resulting in blinking events suitable for reconstructing a super-resolution image (Supporting information, Figure S8). Blinking events were recorded on the same TIRF microscope and analyzed with the Picasso software package.31 After drift correction, the reconstructed image showed individual spots from each monomer with 25 nm full width at half maximum (Figure 3b) corresponding to a localization precision32 of ~10 nm (Figure 3 and Supporting Information, Figure S9). From this, we infer that the DNA structures are immobilized at a scale below our detection limit and thus are suitable frames for super-resolution imaging, especially when they assemble into hexagonal lattices which could offer a high density of attachment sites.24 With this behavior in mind, we designed a scheme for stop-and-go super-resolution imaging to follow the assembly of DNA origami lattices (Figure 4a). First, in the presence of ‘mobility buffer’, which contains 5 mM MgCl2 and 150 mM NaCl, the monomers diffused freely. Addition of the trimerization oligonucleotides (100 nM) triggered the formation of the triskelia. After overnight incubation, we exchanged the buffer to the imaging buffer containing 10 nM of DNA-PAINT imager strands. After drift correction, we found groups of three spots forming triangles indicating successful triskelion formation (Figure 4b). To quantify the quality of these assemblies, we measured the nearest neighbor distance for every spot (Figure 4b, c) and obtained an average nearest neighbor distance of 106 ± 0.7 nm (Supporting Information, Note S1). This value matches the expected distance of 106 nm between neighboring spots in a planar triskelion (Supporting Information, Figure S10). The number of monomers in each assembled structure was determined by counting the spots in the reconstructed image (Figure 4d). While 30% of the assemblies had three monomers, 28% of structures had two monomers, 17 % were single spots and 26% contained more than three spots. This result is actually in opposition to


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the in-solution assembly where most of the miss-assembled and aggregated structures remain in the agarose gel pocket due to their large size (Supporting Information, Figure S2). Importantly, the yield of successfully folded trimers assembled without membrane support in solution was only 10% as assessed by gel electrophoresis (Figure 1b, Supporting Information, Figure S2, Table S3). We thus conclude that assembling these structures confined to two dimensions and with defined orientation – the cholesterol-modified oligonucleotides anchor the monomers with their ‘bottom’ face to the membrane – significantly enhances the yield by favoring correct encounters of the units and by preventing 3D cluster formation. In the next step, we assembled the triskelion structures into hexagonal lattices by adding multimerization oligonucleotides to the mobility buffer followed by overnight incubation. The super-resolved images recorded in imaging buffer on the next day revealed the formation of hexagonal assemblies spanning areas of more than 1 µm2. Hexagonal arrangements are predominant in these images, but also other geometries become apparent. Notably, the high resolution achieved with DNA-PAINT imaging enabled us to resolve the two spots at the connection between the arms of adjacent triskelia (Figure 4e, green circle). By design, these spots have a distance of 18 nm, which is matched very well by our measured distance of 20 nm (Supporting Information, Figure S10 and S11). Additionally, the number of blinking events23 in each spot was similar to that of the monomer (Supporting Information, Figure S12) with the same number of PAINT handles. We further quantified the yield of assembly by measuring the number of monomers in each cluster (Figure 4f). To increase the assembly yield, we also used triskelion structures assembled in solution and purified from agarose gel.33 (Figure 4g). As in the latter case only correctly formed triskelia are present, larger and better-ordered lattices can grow. With multiple stop-and-go cycles we were able to follow the growth of the hexagonal lattice over time on the same membrane revealing intermediate assembly stages (Supporting Information, Figure S13).

Discussion It is known that in the presence of divalent ions, DNA molecules adsorb to lipid membranes with zwitterionic headgroups.18 Notably, if we assume that DNA nanostructures undergo diffusion together with the lipid molecules underneath, their diffusion coefficient should be around 0.1 μm2/s.34, 35 This value fits very well with the slow population of the 1-layer structure


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on the SLBs on mica which is dominant at 0 mM NaCl, indicating the possibility of coupled diffusion of the DNA structures with adsorbed lipid molecules underneath. Although we identified conditions at which our DNA nanostructures stop diffusing on the lipid bilayers, the molecular mechanism for this transient immobilization remains unclear. Our stop-and-go protocol works for several lipid compositions which suggests that the mechanism should not depend on the particular choice of lipid (Supporting information, Figure S14). Woo & Rothemund presented a study in which they followed the assembly of 2D DNA origami lattices on mica surfaces with AFM. This work already revealed a complex interplay of negatively charged DNA, the negatively charged mica and counterions that were present in the buffer.26 The presence of zwitterionic lipid molecules between our substrates and the DNA structures further complicates this issue. From our data, it is clear that magnesium ions slow down or even stop the diffusion completely while sodium ions tend to keep the structures mobile. Previously observed changes of the diffusion coefficient of lipid molecules upon removal of NaCl (two-fold increase)36 or increase of divalent ion (33% decrease)37 solely cannot explain this behavior. Divalent ions like magnesium can cause charge inversion of glass (at 100 mM)38 or DNA (at 1 M)39 both of which are usually negatively charged. Therefore, at certain concentrations of magnesium where charge inversion occurs only at the glass surface, electrostatic interaction between positively charged SiO2 and negatively charged DNA could mediate their binding.40 Thus, one possible reason for the immobilization of DNA structures could be direct charge interaction between the glass surface and the DNA origami objects. In this case, however, no lipid molecules could reside between the DNA and the glass. In our experiments we could neither prove nor disprove this hypothesis. AFM imaging of the lipid membrane revealed a reduction in the membrane thickness when MgCl2 was added (Supporting information, Figure S15). This effect has been observed previously and has been interpreted to reflect a stronger lateral interaction between lipid molecules due to ion-lipid interactions.41, 42 This could to some extent account for the observed slowing down of the DNA objects, but it does not suffice to explain their complete halt. Alternatively, roughness of the glass surface could affect the diffusional behavior of DNA nanostructures and stop their diffusion. For example, when lipid molecules are strongly bound to the DNA nanostructures, the compounds should still be able to move on flat areas but not in regions with strongly deformed bilayers as the rigidity of the structures impedes the adjustment of their curvature. Notably, we found the pre-treatment and hence the roughness of the glass surface to be critical for our observations. If the roughness-reducing KOH treatment was omitted in our substrate cleaning protocol, we


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observed high fractions of immobile DNA objects at all salt conditions (Supporting information, Figure S16). Finally, sub-micrometer defects of the membranes could be responsible for the observed behavior. Such small and possibly transient holes in the bilayers would not be noticeable in FRAP experiments and AFM imaging. In summary, we introduced a method to transiently slow down and arrest DNA origami structures on lipid membranes. Immobilization of DNA origami structures enables the observation of origami lattice formation with super-resolution microscopy. With recently developed 3D super-resolution techniques,43,

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our approach could be generalized to also

image 3D objects on membranes. As our hexagonal lattice resembles clathrin protein complexes on membranes, our method has a potential to study the molecular and physical mechanism of vesicular formation.28 Moreover, this approach can be used to control the diffusional behavior of membrane-targeting molecules when attached to DNA origami structures as cargo molecules. This way it would be possible to study, for example, membrane proteins alternatingly in a mobile and an immobile state. This could shed light on the function of membrane domains and membrane protein complexes. Methods Design, folding and purification of DNA nanostructures DNA origami structures were designed with CaDNAno 2.0 and confirmed by CanDo simulation. Staple oligos generated from the CaDNAno for the structure were purchased from Eurofins Genomics (Ebersberg, Germany). Fluorescence modified oligonucleotides were purchased from Eurofins Genomics while cholesterol-TEG modified oligonucleotides (GGTAGTAATAGGAGAATG 3’ cholesteryl TEG) were purchased from Biomers (Ulm, Germany). Staple oligos were mixed in 100 nM of final concentration with 10 nM of P7650 scaffold. Fluorescent oligos (ATGTAGGTGGTAGAGGAA 3’ Atto 647N) were added at 1 μM concentration if necessary. Structures were annealed for 16 hours starting with a 5 minutes at 65°C denaturing step in the presence of 1X TE and 16 mM of MgCl2.10 After the folding, structures were run in for 2 hours in 2% agarose gel pre-stained with SYBR safe (Thermofisher, USA). Then the structures were purified with Freeze 'N Squeeze™ DNA Gel Extraction Spin Columns from Biorad (Hercules, USA) in mobile buffer. TEM imaging Purified DNA origami structures were imaged using a JEM-1011 transmission electron


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microscope (JEOL). DNA origami structures were attached on argon plasma-exposed (24 W for 1 min) Formvar/carbon-coated grids, which were then negatively stained with 1% uranyl formate for 15 seconds. Measuring diffusion coefficient of DNA nanostructures on supported lipid bilayer 1 mg of 18:1 (Δ9-Cis) PC (DOPC, from Avanti Polar Lipids, Alabaster, USA) dissolved in chloroform was transferred to a clean glass vial using glass syringe from Hamilton (Reno, USA). When membrane integrity or fluidity should be checked, 0.1% (m/m) of NBD C6 HPC was added to DOPC, which revealed that the membrane was fluid in all conditions (data not shown). Then the lipid solution dried out by blowing nitrogen gas to evaporate chloroform. The lipid film was further dried for at least 4 hours under vacuum. Then the lipid film was rehydrated by adding 1 ml of mobile buffer (1×TE, 150 mM NaCl, 5 mM MgCl2) and vortexing well. Small unilamellar vesicles (SUV) were formed by sonicating the lipid solution until it becomes completely clear (~ 20min). This SUV solution was added with final concentration of 0.1mg/ml in a channel of sticky-Slide VI 0.4 from ibidi (Martinsried, Germany). A 1.5# cover glass (60 mm × 20 mm, Carl Roth, Germany) for the channel was cleaned with 2% hellmann (Hellma, Müllheim, Germany) for 20 minutes and further cleaned with 1 M KOH for 40 minutes. Alternatively, a thin layer of freshly cleaved mica was deposited on a droplet of immersion oil on a 1# cover glass (20 mm × 20 mm,). A bottom cut inverted 0.5 mL Eppendorf was UV glued on top of the mica sheet to form a chamber. After incubating 30 minutes, the SUV solution was washed away two times with water and mobile buffer. Then 10 nM of the cholesterol-TEG modified anchor DNA oligonucleotide (GGTAGTAATAGGAGAATG 3’ cholesterol–TEG) was added and incubated for 30 minutes. After washing excess cholesterol oligos with mobile buffer, gel-purified DNA nanostructures with complementary handle for anchor oligo was added to the channel at a final concentration of 0.5 nM. Then the structures were imaged with IX71 inverted microscope from Olympus (Tokyo, Japan) under green illumination with 100 ms of exposure time. Movies were analyzed with MosaicSuite plugin (MPI-CBG, Dresden Germany) in ImageJ. The buffer was exchanged 5 times to measure the diffusion coefficient of the DNA nanostructures under each salt condition. Assembly of triskelion and hexagonal lattice structures on SLB Trimerization oligos (Table S1) were added at a final concentration of 100 nM with mobile buffer inside the channel. After overnight incubation, excess trimerization oligos were washed


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away and the buffer was exchanged for immobilization buffer (1X TE, 20 mM MgCl2). Then imager oligo (CTA GAT GTA T 3’ Atto 655) was added into the channel at a final concentration of 10 nM for DNA-PAINT imaging. For DNA-PAINT imaging, Atto-labelled staple strands were replaced with unlabeled ones during the folding to maximize DNA-PAINT resolution. The movie was taken from home-built objective type TIRF microscope with 660 nm laser (Toptica, Germany) illumination and then analyzed with the ‘Picasso’ software package.31 A detailed protocol for reconstructing super-resolution images using Picasso can be found in the same reference. In brief, each blinking event was identified with ‘min. net gradient’ value in the Picasso adjusted to detect every blinking event. Each event was fitted with a 2D Gaussian using the “maximum likelihood estimation” method. Then the drift during the acquisition was corrected with the “redundant cross-correlation” method followed by 3~5 rounds of spot-based drift correction. The final reconstructed images were rendered with a hot color map and one-pixel blur setting. For hexagonal lattice assembly, buffer was exchanged to mobile buffer and multimerization oligos (Table S1) were added at a final concentration of 100 nM into the channel. After overnight incubation, excess oligos were washed away and the lattices were imaged with DNA-PAINT in the presence of 6 nM of imager oligo after buffer exchange to immobilization buffer.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Strand routing diagram of the structures, diffusion coefficient DNA structures from additional conditions, exemplary traces and images, additional data and sequence of staple strands. Corresponding Author *E-mail: [email protected] Acknowledgments W.B, S.K and T.L acknowledge the support from the European Commission through the ERC grant agreement n° 336440, ORCA and the DFG through the SFB1032 (A6). A.K acknowledges the support of the Graduate School of Quantitative Biosciences Munich. M.T.S. acknowledges support from the International Max Planck Research School for Molecular and Cellular Life Sciences (IMPRS-LS).


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References (1) Rothemund, P. W. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297-302. (2) Douglas, S. M.; Dietz, H.; Liedl, T.; Hogberg, B.; Graf, F.; Shih, W. M. Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes. Nature 2009, 459, 414-418. (3) Dietz, H.; Douglas, S. M.; Shih, W. M. Folding DNA into Twisted and Curved Nanoscale Shapes. Science 2009, 325, 725-730. (4) Voigt, N. V.; Torring, T.; Rotaru, A.; Jacobsen, M. F.; Ravnsbaek, J. B.; Subramani, R.; Mamdouh, W.; Kjems, J.; Mokhir, A.; Besenbacher, F.; Gothelf, K. V. Single-Molecule Chemical Reactions on DNA Origami. Nat. Nanotechnol. 2010, 5, 200-203. (5) Castro, C. E.; Kilchherr, F.; Kim, D. N.; Shiao, E. L.; Wauer, T.; Wortmann, P.; Bathe, M.; Dietz, H. A Primer to Scaffolded DNA Origami. Nat. Methods 2011, 8, 221-229. (6) Langecker, M.; Arnaut, V.; Martin, T. G.; List, J.; Renner, S.; Mayer, M.; Dietz, H.; Simmel, F. C. Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures. Science 2012, 338, 932-936. (7) Czogalla, A.; Petrov, E. P.; Kauert, D. J.; Uzunova, V.; Zhang, Y.; Seidel, R.; Schwille, P. Switchable Domain Partitioning and Diffusion of DNA Origami Rods on Membranes. Faraday Discuss. 2013, 161, 31-43; discussion 113-150. (8) Suzuki, Y.; Endo, M.; Yang, Y.; Sugiyama, H. Dynamic Assembly/Disassembly Processes of Photoresponsive DNA Origami Nanostructures Directly Visualized on a Lipid Membrane Surface. J. Am. Chem. Soc. 2014, 136, 1714-1717. (9) Suzuki, Y.; Endo, M.; Sugiyama, H. Lipid-Bilayer-Assisted Two-Dimensional Self-Assembly of DNA Origami Nanostructures. Nat. Commun. 2015, 6, 8052-8060. (10) Kocabey, S.; Kempter, S.; List, J.; Xing, Y.; Bae, W.; Schiffels, D.; Shih, W. M.; Simmel, F. C.; Liedl, T. Membrane-Assisted Growth of DNA Origami Nanostructure Arrays. ACS Nano 2015, 9, 3530-3539. (11) Bell, N. A.; Keyser, U. F. Digitally Encoded DNA Nanostructures for Multiplexed, Single-Molecule Protein Sensing with Nanopores. Nat. Nanotechnol. 2016, 11, 645-651. (12) Burns, J. R.; Seifert, A.; Fertig, N.; Howorka, S. A Biomimetic DNA-Based Channel for the LigandControlled Transport of Charged Molecular Cargo across a Biological Membrane. Nat. Nanotechnol. 2016, 11, 152-156. (13) Avakyan, N.; Conway, J. W.; Sleiman, H. F. Long-Range Ordering of Blunt-Ended DNA Tiles on Supported Lipid Bilayers. J. Am. Chem. Soc. 2017, 139, 12027-12034. (14) Czogalla, A.; Kauert, D. J.; Franquelim, H. G.; Uzunova, V.; Zhang, Y.; Seidel, R.; Schwille, P. Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles. Angew. Chem. Int. Ed. Engl. 2015, 54, 6501-6505. (15) Khmelinskaia, A.; Franquelim, H. G.; Petrov, E. P.; Schwille, P. Effect of Anchor Positioning on Binding and Diffusion of Elongated 3d DNA Nanostructures on Lipid Membranes. J. Phys. D: Appl. Phys. 2016, 49, 194001-194011. (16) Franquelim, H. G.; Khmelinskaia, A.; Sobczak, J. P.; Dietz, H.; Schwille, P. Membrane Sculpting by Curved DNA Origami Scaffolds. Nat. Commun. 2018, 9, 811-820. (17) Grome, M. W.; Zhang, Z.; Pincet, F.; Lin, C. Vesicle Tubulation with Self-Assembling DNA Nanosprings. Angew. Chem. Int. Ed. Engl. 2018, 57, 5330-5334. (18) Gromelski, S.; Brezesinski, G. DNA Condensation and Interaction with Zwitterionic Phospholipids Mediated by Divalent Cations. Langmuir 2006, 22, 6293-6301. (19) Johnson-Buck, A.; Nangreave, J.; Jiang, S.; Yan, H.; Walter, N. G. Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional DNA Nanostructures. Nano Lett. 2013, 13, 2754-2759. (20) Rust, M. J.; Bates, M.; Zhuang, X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (Storm). Nat. Methods 2006, 3, 793-795. (21) Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006, 313, 1642-1645. (22) Hell, S. W. Microscopy and Its Focal Switch. Nat. Methods 2009, 6, 24-32. (23) Jungmann, R.; Avendano, M. S.; Dai, M.; Woehrstein, J. B.; Agasti, S. S.; Feiger, Z.; Rodal, A.; Yin, P. Quantitative Super-Resolution Imaging with Qpaint. Nat. Methods 2016, 13, 439-442. (24) Dai, M.; Jungmann, R.; Yin, P. Optical Imaging of Individual Biomolecules in Densely Packed Clusters. Nat. Nanotechnol. 2016, 11, 798-807. (25) Lippincott-Schwartz, J.; Snapp, E.; Kenworthy, A. Studying Protein Dynamics in Living Cells. Nat. Rev. Mol. Cell Biol. 2001, 2, 444-456. (26) Woo, S.; Rothemund, P. W. Self-Assembly of Two-Dimensional DNA Origami Lattices Using Cation-


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Design and folding of DNA triskelion. (a) Schematic of DNA triskelion assembly from monomers and its further multimerization into a hexagonal lattice. (b) TEM images of purified monomer (left) and triskelion (right). These triskelia were assembled in solution. All scale bars: 100 nm.


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Diffusion of DNA nanostructures on SLBs at different salt concentrations. (a) Schematic of a triskelion monomer on an SLB. To determine the diffusion coefficient, Atto 647N-modified DNA nanostructures were individually tracked and the traces were analyzed with the ImageJ plugin MosaicSuite. An exemplary trace of a monomer structure diffusing on an SLB on glass is shown at the bottom. Scale bar: 5 μm. (b) The diffusion coefficients of monomer structures on SLB on glass at different salt concentrations showing a bimodal distribution. In buffer containing 20 mM MgCl2 and 0 mM NaCl, all structures stop diffusing (black line). Note that a diffusion coefficient of 10-4 μm2/s corresponds to fluctuations of 102 nm2/s, which is the limit of our tracking accuracy. (c) Diffusion coefficient of 1-layer origami sheet on SLB on glass in different salt concentrations. Most structures do not diffuse in buffers containing 0 mM NaCl and 5 mM MgCl2. (d-e) The diffusion coefficient of the triskelion monomers (d) and the 1-layer sheet (e) on SLB on mica show multimodal distributions. The diffusion slows down at 0 mM NaCl and 5 mM MgCl2 but the objects do not come to halt.


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DNA-PAINT on immobilized DNA nanostructures. (a) Scheme for DNA-PAINT imaging of origami monomer on glass-supported SLB. Three 9 nt-long staple extensions act as docking sequences for transient binding of dye-labeled imager strands. (b) Exemplary super-resolution image of immobilized monomer structures at 0 mM NaCl and 20 mM MgCl2. The full width at half maximum of this localized spot is 25 nm corresponding to a localization precision of ~10 nm. Scale bar: 500 nm.


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Stop-and-go assembly and DNA-PAINT imaging of triskelion structures and hexagonal lattices. (a) Stop-andgo assembly scheme. By changing the salt concentration, DNA nanostructures can either diffuse for higher order assembly or be immobilized for DNA-PAINT imaging. (b) DNA-PAINT image of triskelion structures assembled on glass SLB. Scale bar: 1 μm. (c) Nearest neighbor distance distribution and its Gaussian fit center at 106 ± 0.7 nm. (d) Number of monomers in a cluster after overnight trimerization. (e) DNA-PAINT image of hexagonal lattices formed from membrane-assembled trimers (three-step-assembly) over the course of 3 days. Two spots can be identified where two arms are connected (green circle in inset). Scale bar: 1 μm. (f) Number of monomers in lattice pieces formed from membrane-assembled triskelia. (g) Number of monomers in lattice pieces formed from gel purified triskelia (two-step assembly).


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Single Particle Tracking and Super-Resolution ... - ACS Publications

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