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Stiffness and Membrane Anchor Density Modulate DNA-Nanospring-Induced Vesicle Tubulation
- Michael W GromeMichael W GromeDepartment of Cell Biology, Yale University School of Medicine, and Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, Connecticut 06516, United StatesMore by Michael W Grome
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- Zhao ZhangZhao ZhangDepartment of Cell Biology, Yale University School of Medicine, and Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, Connecticut 06516, United StatesMore by Zhao Zhang
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- Chenxiang Lin*Chenxiang Lin*Email: [email protected]Department of Cell Biology, Yale University School of Medicine, and Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, Connecticut 06516, United StatesMore by Chenxiang Lin
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Abstract
DNA nanotechnology provides an avenue for the construction of rationally designed artificial assemblages with well-defined and tunable architectures. Shaped to mimic natural membrane-deforming proteins and equipped with membrane anchoring molecules, curved DNA nanostructures can reproduce subcellular membrane remodeling events such as vesicle tubulation in vitro. To systematically analyze how structural stiffness and membrane affinity of DNA nanostructures affect the membrane remodeling outcome, here we build DNA-origami curls with varying thickness and amphipathic peptide density, and have them polymerize into nanosprings on the surface of liposomes. We find that modestly reducing rigidity and maximizing the number of membrane anchors not only promote membrane binding and remodeling but also lead to the formation of lipid tubules with better defined diameters, highlighting the ability of programmable DNA-based constructs to controllably deform the membrane.
Introduction
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Intracellular space is full of intricate and dynamic membrane architectures that segregate biochemical reactions and facilitate cellular metabolism. (1,2) Tubular shapes are found as common intermediates during membrane remodeling and are often maintained by well-organized, however transient, protein complexes. (3−9) Creating biomimetic constructs that recapitulate membrane dynamics, such as tubulation in a cell-free environment, is a powerful approach to the mechanistic understanding of protein-mediated membrane remodeling. Further, controllable membrane manipulating techniques may enable the development of drug delivery and cell reprogramming tools. Structural DNA nanotechnology, which takes advantage of the defined DNA structures and base-pairing rules to build nanoscale objects with customizable geometry and chemically addressable surfaces, (10−15) presents an opportunity to dynamically shape membranes. (16−23) For example, a number of studies showed that DNA nanostructures modified with membrane anchors (e.g., cholesterol, amphipathic peptides) were able to drive lipid tubule formation on artificial membranes, including supported lipid bilayers (20) as well as large and giant unilamellar vesicles (LUVs and GUVs). (21−23) Besides proving the concept, this body of work also provides general guidelines for the design (e.g., curvature and membrane anchor placement) and deployment (e.g., osmolality, membrane surface coverage, etc.) of the membrane-sculpting DNA structures. However, the curved DNA structures used in previous studies to imitate membrane-deforming proteins (e.g., BAR and ESCRT) featured high structural rigidity, yet the resulting lipid tubules were somewhat polydispersed in diameter (i.e., curvature). (22,23) This prompted us to study the correlation between the stiffness of DNA structures and their vesicle-tubulation products.
Mechanical properties of multihelix bundle DNA nanostructures greatly depend on the shape and area of their cross-sections. (24,25) Increasing the number of helices in a DNA-origami rod can lead to a quadratic increment in its persistence length. (26,27) We recently built membrane-anchor decorated DNA-origami 24 helix bundle (24HB) curls that form spring-like filaments (DNA nanosprings) on vesicles in response to the addition of linker strands, generating nanospring-scaffolded membrane tubules. (23) Using similar design principles, here we constructed thinner 6HB and 12HB DNA curls that can polymerize into left-handed nanosprings with helical pitches (53 and 54 nm) and diameters (26 and 22 nm) nearly identical to those of 24HB nanosprings (Figure 1a and Figures S1 and S2). Additionally, we outfitted the interior of each monomeric DNA curl (approximately three-quarters of a turn, 100 nm contour length) with a maximum of 24 (24HB) or 16 (12HB and 6HB) single-stranded DNA “handles” for the attachment of amphipathic peptides (adapted from the N-terminus α-helix motif of snf7) as membrane anchors (Figure 1b). (23,28) We expect the three types of nanosprings to have distinct stiffness and tunable membrane anchor density, thus allowing for a systematic investigation of the DNA structures’ membrane binding and tubulation behaviors.
Figure 1
Figure 1. Designs and assembly of DNA-origami nanosprings of varying thicknesses. (a) Cartoon models depicting the 24, 12, and 6 helix-bundle (HB) DNA curls and their linker-triggered polymerization into nanosprings. Each DNA double-helix and DNA-conjugated amphipathic peptide (16 per monomer) are represented by a curved gray rod and a purple sphere, respectively. Negative-stain TEM images of peptide-labeled DNA curls and nanosprings are located next to the corresponding schematics. Scale bars = 100 nm. (b) Top views of DNA-curl models (top) and schematic drawings of the Cy5-modified DNA-peptide conjugate that decorate the interiors of the DNA curls (bottom). (c) An agarose gel resolving DNA curls with varying numbers of inward-facing ssDNA handles (Handle #) in the presence and absence of Cy5-modified antihandle-peptide (AH-peptide) conjugates. Bands that contain monomeric DNA curls and misfolded aggregates are boxed and labeled with asterisks (*) and daggers (†), respectively. Pseudocolor fluorescence: red = Cy5, green = ethidium bromide.
Results and Discussion
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We first examined the assembly and labeling of the DNA nanosprings in aqueous solutions. Negative-stain transmission electron microscopy (TEM) showed that the purified, well-folded DNA curl monomers exhibited contour lengths (24HB: 101 ± 4 nm, 12HB: 81 ± 5 nm, and 6HB: 81 ± 5 nm), thicknesses (24HB: 13 ± 1 nm, 12HB: 8 ± 1 nm, and 6HB: 7 ± 1 nm), and linker-strand-triggered polymerization capability in accordance with the design (Table S1 and Figure S3). All three types of nanosprings showed comparable helical pitches (24HB, 80 ± 9 nm; 12HB, 70 ± 5 nm; and 6HB, 75 ± 5 nm) that are larger than the designed values, likely due to the global left-handed twisting not accounted for by our bending model (29) and the DNA structures splaying on TEM grids. The inner diameters of 24HB, 12HB, and 6HB springs measured 26 ± 5 nm, 16 ± 6 nm, and 8 ± 2 nm, respectively, suggesting DNA filaments with smaller cross sections were more prone to structural distortion (Table S2 and Figure S3). The increased flexibility associated with thinner DNA filaments is qualitatively corroborated by the measured persistence lengths of the nanosprings (Table S3). We note that the DNA springs’ stiffness can also be reduced by self-assembly errors that occur during the folding and polymerization of the DNA curls. (30,31) Hybridizing Cy5-modified DNA-peptide conjugates to the inner handles of DNA curls yielded mostly monomeric DNA structures with Cy5 signal proportional to the number of handles (0, 7, 16, or 24) per monomer, as confirmed by nondenaturing agarose gel electrophoresis (Figure 1c and Figure S4).
Next, we tested the binding affinity of the 24HB, 12HB, and 6HB DNA curls (4 nM) with 0, 7, or 16 peptides toward rhodamine-labeled LUVs (39.7 μM 1,2-dioleoyl-sn-glycero-3-phosphocholine or DOPC, 0.3 μM 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) or Rhod-PE), extruded through a polycarbonate membrane with a pore size of 200 nm (mean vesicle diameter: ∼134 nm, determined by TEM). This 10 000:1 lipid-to-curl ratio translates to ∼15 curls per average-sized LUV (consisting of ∼150 000 lipid) in a hypothetical scenario where all DNA curls were associated with membrane. Using our previously established protocol, (23) the DNA curls and vesicles were incubated for 1 h at 4 °C in 10 mM MgCl2 and 400 mM KCl buffer solution to allow DNA structures to bind to membrane without changes in osmolarity (Figure 2a). Immediately following, mixtures underwent an isopycnic centrifugation to isolate the LUV-bound DNA structures from the unbound ones. Fluorescence scans revealed that stronger lipid-DNA colocalization is generally associated with increasing number of peptide labels and decreasing DNA-curl thickness (Figure 2b, with the complete data set and quantification shown in Figure S5). This can be explained by the better accessibility of the peptides on the thinner DNA curls, consistent with the notion that membrane anchors can be sterically blocked by bulky DNA-origami structures. (32,33) Additionally, the flexibility to “breath” may augment the readiness of the less stiff curls to interact their amphipathic interiors with membranes of different curvatures and tensions. A negative-stain TEM study on unpurified mixtures of DNA curls and LUVs corroborated the centrifugation results. Here, thinner DNA curls labeled with more peptides left noticeably fewer unbound monomers on the TEM grids; in all TEM specimens there were no apparent reduction in the concentration of LUVs to suggest DNA-curl induced aggregation. Interestingly, only 12HB and 6HB curls carrying 16 peptides per monomer led to tubule formation after 1 h of incubations in the absence of linker strands (Figure S6). This positive correlation between membrane affinity and linker-independent vesicle tubulation is consistent with previous findings that high membrane coverage by curved DNA structures is a prerequisite for vesicle tubulation. (22,23)
Figure 2
Figure 2. Large unilamellar vesicle (LUV) binding and tubulation activities of self-assembling DNA curls of varying thicknesses and membrane anchor densities. (a) Schematic illustration of the experiment procedures used to test DNA-curl induced LUV tubulation with and without linkers. (b) Fractionated density gradients containing Cy5-labeled DNA curls with 16 or 0 amphipathic peptides per monomer (Pep #) and rhodamine-labeled LUVs after isopycnic centrifugation. Pseudocolor fluorescence of recovered fractions: red = Cy5, blue = rhodamine. Smaller numbers denote lighter fractions. (c) Normalized abundances of lipid tubules generated by 24, 12, or 6 helix-bundle (HB) curls with 16 or 7 peptides per monomer in the presence or absence of linker. For each measurement, 750–2000 vesicles were surveyed. (d) Representative negative-stain TEM images of LUVs tubulated by DNA curls (16 peptide per monomer) of different thicknesses, with and without DNA linkers. Scale bars = 400 nm.
After the initial binding tests at 4 °C for 1 h, we held the mixture of vesicles and DNA curls at 30 °C for another 16 h in the presence or absence of linker strands (Figure 2a) and analyzed the resulting lipid tubules using negative-stain TEM. In general, without linker strands, tubulation efficiency of DNA curls positively correlated with their membrane binding affinity (Figure 2c and Figures S5 and S7–S9). Namely, all three types of curls labeled with 16 peptides generated appreciable amounts of tubules, with more flexible structural designs resulting in higher tubulation efficiency (24HB, 2.7%; 12HB, 6.5%; 6HB, 8.5%; quantified as number of tubules/number of vesicles), whereas their 7-peptide-labeled counterparts generated little to no tubules (24HB, 0.3%; 12HB, 1.2%; 6HB, 0.6%). When the DNA coatings were discernible on the electron micrographs, we found that these lipid tubules were densely covered by irregular patterns of DNA curls. In contrast, having linker strands during the extended incubation period induced the formation of nanosprings and improved tubulation efficiency in all cases (Figure 2c, d). For example, the 16-peptide-labeled 24HB, 12HB, and 6HB nanosprings led to tubulation efficiency of ∼7.1, ∼11.1, and ∼9.0%, respectively. However, polymerizing handle-less DNA curls with free peptides did not generate tubulation (Figure S10). Surprisingly, polymerized 12HB curls produced more tubules than the 6HB curls, despite the latter being stronger LUV binders in the monomeric form. We initially speculated that this is because the linker strands promote tubulation partially by recruiting additional DNA-curl monomers to the membrane; thus, tubulation by stiffer springs benefits from a larger supply of free monomers left over in solution after the initial binding. This also seemed to explain the larger boost in tubulation efficiency when stiffer DNA curls were polymerized on membrane—24HB, 12HB, and 6HB curls equipped with 16 peptides produced 160, 71, and 5.8% more tubules with linker strands added, respectively. However, this hypothesis does not explain the tubulation efficiencies of 7-peptide-labeled structures, where the thinner, more lipophilic curls saw a larger tubulation boost upon polymerization (Figure S11). Therefore, it seemed that all curls must reach certain surface coverage for membrane remodeling to happen efficiently, (22,23) and once this requirement is met, stiffer structures may have advantages in tubulation. For example, with 7 or 16 peptides per monomer, tubulation by the 24HB curl was inferior to that by the 12HB and 6HB curls because of its poor membrane affinity. However, modifying each monomer with 24 peptides brought 24HB curl’s membrane coverage to a level comparable to the 7-peptide-labeled 12HB and 6HB curls; under this condition, the 24HB structure produced 4–8 fold more tubes than the 12HB and 6HB curls (Figure S5). Nevertheless, with the same peptide-labeling density, DNA-to-lipid ratio, and polymerizing condition, we found the 12HB curls to be the best-performing membrane-deforming structure. Increasing Mg2+ concentration from 10 to 15 mM further enhanced the 12HB curls’ tubulation efficiency from 11.1 to 15.6% (16 peptide per monomer) and from 4.7 to 10.8% (7 peptide per monomer) as well as producing longer tubules with average lengths beyond 500 nm (Figures S12–S15 and Table S4), likely due to more efficient linker-induced nanospring formation under higher divalent cation concentration.
Although the TEM studies do not permit direct visualization of tubulation kinetics, examining the morphologies of lipid tubules can give clues as to their formation mechanism. For example, although polymerizing 24HB curls on LUVs yielded tubules with considerable width heterogeneity, the tubules with a dense DNA coat generally had widths that conform to the inner diameter of the nanosprings, suggesting that the nanosprings control the tubule width only when sufficient surface density is achieved. (23) Therefore, we expected the 12HB and 6HB nanosprings, because of their thinner filaments and greater compressibility (i.e., smaller spring constant), to cover the membrane surfaces with high density and result in better controlled lipid-tubule diameters. Our analyses on lipid-tubule widths and apparent DNA-nanospring pitches (peak-to-peak distance of the DNA coat) showed that this was indeed the case (Figure 3). Labeled with 16 peptides per monomer, 12HB and 6HB nanosprings produced tubules with diameters of 28.2 ± 4.4 and 27.1 ± 3.6 nm, respectively, which are notably more homogeneous than those produced by the 24HB nanosprings (23.7 ± 7.2 nm) in the same conditions (Figure 3b). The 12HB nanosprings on lipid tubules also showed much smaller and monodispersed apparent pitches (19.2 ± 2.5 nm) relative to 24HB nanosprings (34.9 ± 17.7 nm), consistent with our expectation (Figure 3c). These comparisons held true when peptide labels were reduced to seven per monomer, albeit with overall reduced tube abundance and homogeneity (Figure S16 and Table S4). Enhancing the membrane affinity of 24HB curls by labeling each monomer with 24 peptides and doubling the DNA-to-lipid ratio produced more tubes with widths approximating the inner diameter of the nanosprings. However, the overall tube-width homogeneity was still less than those produced by the 12HB and 6HB springs (Figure S16). High-magnification TEM images revealed that the majority of lipid tubules formed by the 12HB nanosprings were coated by intertwined triplexes of modestly compressed nanosprings, each with a mean helical pitch ≈ 47 nm that translates to an apparent pitch of ∼16 nm. This means such tubules were nearly completely covered by DNA, considering the ∼11 nm designed thickness of the 12HB filaments. By contrast, 24HB nanosprings compressed to a comparable level (mean helical pitch ≈ 42 nm) formed primarily duplexes of ∼21 nm apparent pitches (Figure 4); in fact, most 24HB springs remained uncompressed, leading to a larger percentage of uncovered membrane surfaces (Figure S17). In other words, comparing to the thicker, stiffer 24HB nanosprings, the thinner, more flexible 12HB nanosprings enforced stricter confinement of tubule width by forming a denser DNA-spring coating on lipid tubes as a result of higher membrane affinity, and greater susceptibility to compression and multiplex formation. This also embedded nearly 50% more peptides into the same length of tube (Figure 4), which introduced greater leaflet asymmetry to promote tubulation (Figure S18). Although most of the 6HB filaments did not generate enough contrast under TEM to be measured on membrane, we can extrapolate and expect a dense DNA coating on the lipid tubules, explaining the best uniformity of tube diameter produced by the three types of nanosprings.
Figure 3
Figure 3. Impact of DNA-nanospring thickness on lipid-tubule morphologies. (a) TEM images of lipid tubules covered by 24, 12, and 6 helix-bundle (HB) nanosprings. Scale bars = 100 nm. (b) Average tubule widths as a result of polymerizing 24, 12, and 6HB DNA curls (16 amphipathic peptides per monomer) mixed with LUVs. Means are weighted for the tubule lengths. Error bars show standard deviations. (c) Scatter plots showing the widths of lipid tubules versus the apparent pitches of the tubule-associating 24 and 12HB nanosprings (defined as the length of nanospring-coated tubule divided by the cumulative helical turns of DNA filaments).
Figure 4
Figure 4. Cartoon models and the corresponding TEM images contrasting the compressed 24 and 12 helix-bundle (HB) DNA nanosprings (gray, blue, and red) intertwining around lipid tubules (tan). The measured apparent and actual pitches of nanosprings are labeled at the bottom and top of each model, respectively. The “see-through” box reveals the different surface densities of peptides (purple dots) introduced by the 24 and 12HB nanosprings. Scale bar = 100 nm.
Conclusions
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Nature has evolved membrane-deforming proteins that are not only efficient and dynamic, but also under layers of regulation. (6,7,34,35) When engineering artificial systems to mimic such elegance of nature, it is therefore important to understand the design parameters that modulate the membrane remodeling in vitro. By varying the curvature of a stiff (20HB) DNA structure labeled with cholesterols, Franquelim et al. found that a modest bend led to the most effective GUV tubulation. (22) Here we showed that the filament thickness of DNA nanospring profoundly impacted the frequency of LUV tubulation events and the morphology of the lipid tubules. Clearly, further investigation is needed to fully elucidate the DNA-nanospring-induced membrane deformation mechanism (e.g., by controlling membrane curvature and tension, by monitoring the deformation kinetics). With this in mind, the main contribution of this work is establishing stiffness as a tunable parameter that affects vesicle tubulation outcome and providing a practical guide for designing and choosing from an assortment of “strong” and “weak” DNA nanosprings for membrane deformation. Our findings implied that the abilities to (i) promiscuously bind membranes with various curvatures and (ii) readily propagate into a dense coat of nanosprings on membrane surfaces are key for curved DNA structures to efficiently draw structurally defined lipid tubules from vesicles. This insight will inform the future implementation of more advanced membrane deforming machineries that could, for example, carry specific ligands to target curved biological membranes and constrict membrane tubules to enable scission.
Experimental Section
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Materials
DNA oligonucleotides were purchased from Integrated DNA Technologies (see Supporting Information for sequences). DNA-peptide conjugates were purchased from Bio-Synthesis. Lipids and liposome extrusion filters were purchased from Avanti Polar Lipids. TEM grids and uranyl formate were purchased from Electron Microscopy Sciences. JM109 E. coli cells and VCSM13 helper phage were purchased from Agilent Technologies. Ultracentrifuge tubes were purchased from Beckman Coulter. Iodixanol was purchased from Stemcell Technologies. Other chemicals and Amicon filters were purchased from Millipore Sigma.
DNA-Nanospring Design and Preparation
Three-dimensional models of DNA-origami curls were initially built in Autodesk Maya, which guided the DNA-origami design by caDNAno (cadnano.org). (36) The models and design diagrams are available in Figure S1 and S2. M13-derived scaffold DNA strands (8064 and 7308-nt long) were produced using E. coli and bacteriophages as previously described. (37) The pBlueScript-derived scaffold (3024-nt long) was prepared with JM109 cells and VCSM13 using a single-stranded phagemid rescue protocol recommended by the manufacturer. The DNA curls were folded from a mixture of scaffold strand (40 nM) and staple strands (240 nM each) in a TE buffer (pH 8) containing 10 (for 6HB) or 15 mM (for 12 and 24HB) MgCl2 using a 36-h thermal annealing (80–24 °C) program. Correctly folded DNA curls were purified and concentrated by rate-zonal centrifugation and Amicon filtration. (38) To functionalize the DNA structures, purified curls displaying handles were then incubated with peptide or Cy5-modified antihandles at antihandle-to-handle ratio of 3:1. The excessive antihandles were removed by polyethylene-glycol (PEG) fractionation. (39) To form nanosprings, we added linker strands to the peptide/Cy5-labeled DNA curls in 20-fold molar excess; the mixture was incubated at 30 °C for 16 h.
Gel Electrophoresis
Equal molar amount of DNA curl monomers (with or without peptides and Cy5 labels) were loaded into separate lanes of a 1.8% agarose gel casted with 0.5 × TBE buffer containing 10 mM MgCl2. The gel was run in the same buffer solution at 5 V/cm for 3 h before being imaged on a Typhoon FLA 9500 scanner using laser and filter settings for the Cy5-fluorescence channel. It was then stained by ethidium bromide (EtBr), destained, and imaged again using settings for the Cy5 and EtBr channels.
DNA-Curl-Mediated Vesicle Tubulation
LUVs (99.2% DOPC, 0.8% Rhod-PE, 15 mM total lipid) were prepared via lipid-film rehydration followed by extrusion. Unless noted otherwise, these vesicles (final lipid concentration = 40 μM) were mixed with peptide-labeled, PEG-fractionated DNA curls at 10 000:1 lipid-to-curl ratio in a 25 mM HEPES buffer (pH 7.5) containing 400 mM KCl and 10 mM MgCl2, incubated for 1 h at 4 °C (to allow binding), and brought to 30 °C for an 16-h incubation in the presence (20-fold excess, to trigger polymerization on membrane) or absence (for the study of linker-independent tubulation) of linker strands.
Isopycnic Centrifugation
One hundred microliters of preincubated LUVs and DNA curls (prepared as described above) was diluted to a final volume of 300 μL, which consisted of 30% iodixanol, 25 mM HEPES (pH 7.5), 10 mM MgCl2, and 400 mM KCl. This mixture was transferred to a 0.8 mL ultracentrifuge tube, on top of which six layers (60 μL each) of solutions with decreasing iodixanol concentrations (26–6%, with 4% decrement per layer) were added. The content of this tube was spun at 48 000 rpm at 4 °C in a swing bucket rotor (SW 55 Ti) for 5 h, fractionated from top to bottom into 13 wells of a 96-well plate (∼52 μL per fraction), and imaged on a Typhoon FLA 9500 scanner in Cy5 and rhodamine channels.
Transmission Electron Microscopy
A drop (∼5 μL) of sample was deposited on a glow-discharged carbon-coated copper grid and allowed to adsorb for 1–2 min before blotted away. Negative-stain was achieved by adding 2% uranyl formate solution to the grid and incubating for 1–3 min. After blotting away the stain, the grid was air-dried and imaged using a JEOL JEM-1400 plus microscope operated at 80 kV. Images were acquired by a 4k × 3k CCD camera (Advanced Microscopy Technologies) and analyzed by ImageJ (National Institutes of Health) for tubule abundance and dimensions, and EasyWorm (40) for persistence lengths.
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05401.
Designs of DNA-origami curls, DNA sequences, materials and methods, and additional data and models (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
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- Chenxiang Lin - Department of Cell Biology, Yale University School of Medicine, and Nanobiology Institute, Yale University, 850 West Campus Drive, West Haven, Connecticut 06516, United States;
http://orcid.org/0000-0001-7041-1946;
Acknowledgments
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The authors acknowledge a National Institutes of Health (NIH) Director’s New Innovator Award (DP2-GM114830) and a Yale University faculty startup fund to C.L. as well as an NIH training grant (T32-GM007499) that supports M.W.G.
References
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This article references 40 other publications.
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- 3Bassereau, P.; Jin, R.; Baumgart, T.; Deserno, M.; Dimova, R.; Frolov, V. A.; Bashkirov, P. V.; Grubmuller, H.; Jahn, R.; Risselada, H. J.; Johannes, L.; Kozlov, M. M.; Lipowsky, R.; Pucadyil, T. J.; Zeno, W. F.; Stachowiak, J. C.; Stamou, D.; Breuer, A.; Lauritsen, L.; Simon, C.; Sykes, C.; Voth, G. A.; Weikl, T. R. The 2018 Biomembrane Curvature and Remodeling Roadmap. J. Phys. D: Appl. Phys. 2018, 51, 343001, DOI: 10.1088/1361-6463/aacb98Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFKrtbrI&md5=e3f07db0ce64c6a12c5ec6063fcab37bThe 2018 biomembrane curvature and remodeling roadmapBassereau, Patricia; Jin, Rui; Baumgart, Tobias; Deserno, Markus; Dimova, Rumiana; Frolov, Vadim A.; Bashkirov, Pavel V.; Grubmuller, Helmut; Jahn, Reinhard; Risselada, H. Jelger; Johannes, Ludger; Kozlov, Michael M.; Lipowsky, Reinhard; Pucadyil, Thomas J.; Zeno, Wade F.; Stachowiak, Jeanne C.; Stamou, Dimitrios; Breuer, Artu; Lauritsen, Line; Simon, Camille; Sykes, Cecile; Voth, Gregory A.; Weikl, Thomas R.Journal of Physics D: Applied Physics (2018), 51 (34), 343001/1-343001/42CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)A review. The importance of curvature as a structural feature of biol. membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphol. are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.
- 4Jarsch, I. K.; Daste, F.; Gallop, J. L. Membrane Curvature in Cell Biology: An Integration of Molecular Mechanisms. J. Cell Biol. 2016, 214, 375– 387, DOI: 10.1083/jcb.201604003Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFKktLnL&md5=d574a24980a8a7ed5fdd74f430a5037fMembrane curvature in cell biology: an integration of molecular mechanismsJarsch, Iris K.; Daste, Frederic; Gallop, Jennifer L.Journal of Cell Biology (2016), 214 (4), 375-387CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)Curving biol. membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common mol. mechanisms for bending membranes are evident in different cell biol. contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the mol. mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the mol. mechanisms we expect to see used by plants and protists.
- 5Frost, A.; Unger, V. M.; De Camilli, P. The Bar Domain Superfamily: Membrane-Molding Macromolecules. Cell 2009, 137, 191– 196, DOI: 10.1016/j.cell.2009.04.010Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmvVaiurg%253D&md5=c2355b3dbd92094526b075074f376e88The BAR domain superfamily: membrane-molding macromoleculesFrost, Adam; Unger, Vinzenz M.; De Camilli, PietroCell (Cambridge, MA, United States) (2009), 137 (2), 191-196CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.
- 6Hurley, J. H.; Hanson, P. I. Membrane Budding and Scission by the Escrt Machinery: It’s All in the Neck. Nat. Rev. Mol. Cell Biol. 2010, 11, 556– 566, DOI: 10.1038/nrm2937Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotFeltL0%253D&md5=9115205a75cdc132eb475f09871fb308Membrane budding and scission by the ESCRT machinery: it's all in the neckHurley, James H.; Hanson, Phyllis I.Nature Reviews Molecular Cell Biology (2010), 11 (8), 556-566CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. The endosomal sorting complexes required for transport (ESCRTs) catalyze one of the most unusual membrane remodeling events in cell biol. ESCRT-I and ESCRT-II direct membrane budding away from the cytosol by stabilizing bud necks without coating the buds and without being consumed in the buds. ESCRT-III cleaves the bud necks from their cytosolic faces. ESCRT-III-mediated membrane neck cleavage is crucial for many processes, including the biogenesis of multivesicular bodies, viral budding, cytokinesis, and, probably, autophagy. Recent studies of ultrastructures induced by ESCRT-III overexpression in cells and the in vitro reconstitution of the budding and scission reactions have led to breakthroughs in understanding these remarkable membrane reactions.
- 7Ford, M. G.; Mills, I. G.; Peter, B. J.; Vallis, Y.; Praefcke, G. J.; Evans, P. R.; McMahon, H. T. Curvature of Clathrin-Coated Pits Driven by Epsin. Nature 2002, 419, 361– 366, DOI: 10.1038/nature01020Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XntlGgsLc%253D&md5=c4874e53217ae4239c2809e5471c5279Curvature of clathrin-coated pits driven by epsinFord, Marijn G. J.; Mills, Ian G.; Peter, Brian J.; Vallis, Yvonne; Praefcke, Gerrit J. K.; Evans, Philip R.; McMahon, Harvey T.Nature (London, United Kingdom) (2002), 419 (6905), 361-366CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P2 in conjunction with clathrin polymn. We have discovered that the formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.
- 8Roux, A.; Cappello, G.; Cartaud, J.; Prost, J.; Goud, B.; Bassereau, P. A Minimal System Allowing Tubulation with Molecular Motors Pulling on Giant Liposomes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5394– 5399, DOI: 10.1073/pnas.082107299Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKlsLs%253D&md5=c43c4d4592dc899963d857aae7f22ad8A minimal system allowing tubulation with molecular motors pulling on giant liposomesRoux, Aurelien; Cappello, Giovanni; Cartaud, Jean; Prost, Jacques; Goud, Bruno; Bassereau, PatriciaProceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5394-5399CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The elucidation of phys. and mol. mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that mol. motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin mols. have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was obsd. when kinesins were directly bound to the GUV membrane, suggesting that it is crit. to distribute the load on both lipids and motors by means of beads. A kinetic anal. shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled expts. aimed at the understanding of basic mechanisms in intracellular transport.
- 9Takei, K.; Slepnev, V. I.; Haucke, V.; De Camilli, P. Functional Partnership between Amphiphysin and Dynamin in Clathrin-Mediated Endocytosis. Nat. Cell Biol. 1999, 1, 33– 39, DOI: 10.1038/9004Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXksVKitrw%253D&md5=f66e85a3d5fb1a392d3642d5e1520139Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosisTakei, Kohji; Slepnev, Vladimir I.; Haucke, Volker; De Camilli, PietroNature Cell Biology (1999), 1 (1), 33-39CODEN: NCBIFN; ISSN:1465-7392. (Macmillan Magazines Ltd)Amphiphysin, a protein that is highly concd. in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphol. evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.
- 10Seeman, N. C. Nucleic Acid Junctions and Lattices. J. Theor. Biol. 1982, 99, 237– 247, DOI: 10.1016/0022-5193(82)90002-9Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXhtlWhuw%253D%253D&md5=82c15bbb6dadb9b373d9da1a68ba23e3Nucleic acid junctions and latticesSeeman, Nadrian C.Journal of Theoretical Biology (1982), 99 (2), 237-47CODEN: JTBIAP; ISSN:0022-5193.It is possible to generate sequences of oligomeric nucleic acids which will preferentially assoc. to form migrationally immobile junctions, rather than linear duplexes. These structures are predicted on the maximization of Watson-Crick base pairing and the lack of sequence symmetry customarily found in their analogs in living systems. Criteria which oligonucleotide sequences must fulfill to yield these junction structures are presented. The generable junctions are nexuses, from which 3-8 double helices may emanate. Each junction may be treated as a macromol. valence cluster, and individual clusters may be linked together directly, or with pieces of linear DNA interspersed between them. This covalent linkage can be done with enormous specificity, using sticky-ended ligation techniques. It appears to be possible to generate covalently joined 3-dimensional networks of nucleic acids which are periodic in connectivity and perhaps in space.
- 11Rothemund, P. W. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297– 302, DOI: 10.1038/nature04586Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitlKgu7g%253D&md5=583caefdda9b1deb5d3f2ef78d9e6ecbFolding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 12Douglas, 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, DOI: 10.1038/nature08016Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtFKmtbs%253D&md5=9c6876c4b0358a9f31a0f4aaa5ba855aSelf-assembly of DNA into nanoscale three-dimensional shapesDouglas, Shawn M.; Dietz, Hendrik; Liedl, Tim; Hogberg, Bjorn; Graf, Franziska; Shih, William M.Nature (London, United Kingdom) (2009), 459 (7245), 414-418CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase scaffold strand' that is folded into a flat array of antiparallel helixes by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helixes constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concns. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manuf. of sophisticated devices bearing features on the nanometer scale.
- 13Dietz, H.; Douglas, S. M.; Shih, W. M. Folding DNA into Twisted and Curved Nanoscale Shapes. Science 2009, 325, 725– 730, DOI: 10.1126/science.1174251Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptl2guro%253D&md5=8dd007a0fba43e591d45b3640b129f10Folding DNA into Twisted and Curved Nanoscale ShapesDietz, Hendrik; Douglas, Shawn M.; Shih, William M.Science (Washington, DC, United States) (2009), 325 (5941), 725-730CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly crosslinked double helixes, arrayed in parallel to their helical axes. Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quant. controlled, and a radius of curvature as tight as 6 nm was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.
- 14Seeman, N. C.; Sleiman, H. F. DNA Nanotechnology. Nat. Rev. Mater. 2018, 3, 17068, DOI: 10.1038/natrevmats.2017.68Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslOntLbM&md5=a8683cd5a65e013464f37bb6383853b4DNA nanotechnologySeeman, Nadrian C.; Sleiman, Hanadi F.Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
- 15Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584– 12640, DOI: 10.1021/acs.chemrev.6b00825Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1CjsL0%253D&md5=db637726b0112f270fe979339d24f96aDNA Origami: Scaffolds for Creating Higher Order StructuresHong, Fan; Zhang, Fei; Liu, Yan; Yan, HaoChemical Reviews (Washington, DC, United States) (2017), 117 (20), 12584-12640CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. DNA has become one of the most extensively used mol. building blocks for engineering self-assembling materials. DNA origami is a technique that uses hundreds of short DNA oligonucleotides, called staple strands, to fold a long single-stranded DNA, which is called a scaffold strand, into various designer nanoscale architectures. DNA origami has dramatically improved the complexity and scalability of DNA nanostructures. Due to its high degree of customization and spatial addressability, DNA origami provides a versatile platform with which to engineer nanoscale structures and devices that can sense, compute, and actuate. These capabilities open up opportunities for a broad range of applications in chem., biol., physics, material science, and computer science that have often required programmed spatial control of mols. and atoms in three-dimensional (3D) space. This review provides a comprehensive survey of recent developments in DNA origami structure, design, assembly, and directed self-assembly, as well as its broad applications.
- 16Zhang, Z.; Yang, Y.; Pincet, F.; Llaguno, M. C.; Lin, C. Placing and Shaping Liposomes with Reconfigurable DNA Nanocages. Nat. Chem. 2017, 9, 653– 659, DOI: 10.1038/nchem.2802Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCrtb3L&md5=e5e94cb3f83dba3ca54caa3428bcce4cPlacing and shaping liposomes with reconfigurable DNA nanocagesZhang, Zhao; Yang, Yang; Pincet, Frederic; Llaguno, Marc C.; Lin, ChenxiangNature Chemistry (2017), 9 (7), 653-659CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modeling biol. membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here, we report the assembly, arrangement and remodeling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.
- 17Czogalla, 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. 2015, 54, 6501– 6505, DOI: 10.1002/anie.201501173Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslart78%253D&md5=cca84a507f151e29a2a5bf80059c6126Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane VesiclesCzogalla, Aleksander; Kauert, Dominik J.; Franquelim, Henri G.; Uzunova, Veselina; Zhang, Yixin; Seidel, Ralf; Schwille, PetraAngewandte Chemie, International Edition (2015), 54 (22), 6501-6505CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report a synthetic biol.-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions giving ordered arrays on the membrane. Such a tight and regular arrangement makes the authors' DNA origami capable of deforming free-standing lipid membranes, mimicking the biol. activity of coat-forming proteins, for example, from the I-/F-BAR family.
- 18Kocabey, 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, DOI: 10.1021/acsnano.5b00161Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjslemsbc%253D&md5=1599e15c303bf8f52be09c43cf8f4a3cMembrane-Assisted Growth of DNA Origami Nanostructure ArraysKocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M.; Simmel, Friedrich C.; Liedl, TimACS Nano (2015), 9 (4), 3530-3539CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Biol. membranes fulfill many important tasks within living organisms. In addn. to sepg. cellular vols., membranes confine the space available to membrane-assocd. proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. The authors here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors, a three-layered rectangular block and a Y-shaped DNA structure, to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coeffs. depending on their size and no. of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures assocd. into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. The authors' results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.
- 19Howorka, S. Nanotechnology. Changing of the Guard. Science 2016, 352, 890– 891, DOI: 10.1126/science.aaf5154Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVSrs7c%253D&md5=cd1bbdcddd6afb5666d0e21d7713bd51Changing of the guardHoworka, StefanScience (Washington, DC, United States) (2016), 352 (6288), 890-891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)There is no expanded citation for this reference.
- 20Birkholz, O.; Burns, J. R.; Richter, C. P.; Psathaki, O. E.; Howorka, S.; Piehler, J. Multi-Functional DNA Nanostructures That Puncture and Remodel Lipid Membranes into Hybrid Materials. Nat. Commun. 2018, 9, 1521, DOI: 10.1038/s41467-018-02905-wGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjktFKqtg%253D%253D&md5=8f8cbce2240bfd769f91e2bf3900f0f8Multi-functional DNA nanostructures that puncture and remodel lipid membranes into hybrid materialsBirkholz Oliver; Richter Christian P; Psathaki Olympia E; Piehler Jacob; Burns Jonathan R; Howorka StefanNature communications (2018), 9 (1), 1521 ISSN:.Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers. Here we use single-molecule fluorescence microscopy to establish that DNA nanopores carrying cholesterol anchors insert via a two-step mechanism into membranes. Nanopores are furthermore shown to locally cluster and remodel membranes into nanoscale protrusions. Most strikingly, the DNA pores can function as cytoskeletal components by stabilizing autonomously formed lipid nanotubes. The combination of membrane puncturing and remodeling activity can be attributed to the DNA pores' tunable transition between two orientations to either span or co-align with the lipid bilayer. This insight is expected to catalyze the development of future functional nanodevices relevant in synthetic biology and nanobiotechnology.
- 21Gopfrich, K.; Zettl, T.; Meijering, A. E.; Hernandez-Ainsa, S.; Kocabey, S.; Liedl, T.; Keyser, U. F. DNA-Tile Structures Induce Ionic Currents through Lipid Membranes. Nano Lett. 2015, 15, 3134– 3138, DOI: 10.1021/acs.nanolett.5b00189Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlsFaqtL8%253D&md5=88671fefb314500fadfcc611970480cbDNA-tile structures induce ionic currents through lipid membranesGopfrich, Kerstin; Zettl, Thomas; Meijering, Anna E. C.; Hernandez-Ainsa, Silvia; Kocabey, Samet; Liedl, Tim; Keyser, Ulrich F.Nano Letters (2015), 15 (5), 3134-3138CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diam. of 5 nm and a mol. wt. of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biol. ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biol. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.
- 22Franquelim, H. G.; Khmelinskaia, A.; Sobczak, J. P.; Dietz, H.; Schwille, P. Membrane Sculpting by Curved DNA Origami Scaffolds. Nat. Commun. 2018, 9, 811, DOI: 10.1038/s41467-018-03198-9Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MrkvVOlsw%253D%253D&md5=308b629abe07442332c291a889c7a404Membrane sculpting by curved DNA origami scaffoldsFranquelim Henri G; Khmelinskaia Alena; Schwille Petra; Khmelinskaia Alena; Sobczak Jean-Philippe; Dietz HendrikNature communications (2018), 9 (1), 811 ISSN:.Membrane sculpting and transformation is essential for many cellular functions, thus being largely regulated by self-assembling and self-organizing protein coats. Their functionality is often encoded by particular spatial structures. Prominent examples are BAR domain proteins, the 'banana-like' shapes of which are thought to aid scaffolding and membrane tubulation. To elucidate whether 3D structure can be uncoupled from other functional features of complex scaffolding proteins, we hereby develop curved DNA origami in various shapes and stacking features, following the presumable design features of BAR proteins, and characterize their ability for membrane binding and transformation. We show that dependent on curvature, membrane affinity and surface density, DNA origami coats can indeed reproduce the activity of membrane-sculpting proteins such as BAR, suggesting exciting perspectives for using them in bottom-up approaches towards minimal biomimetic cellular machineries.
- 23Grome, M. W.; Zhang, Z.; Pincet, F.; Lin, C. Vesicle Tubulation with Self-Assembling DNA Nanosprings. Angew. Chem., Int. Ed. 2018, 57, 5330– 5334, DOI: 10.1002/anie.201800141Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnt1elsL0%253D&md5=5da4fa7a1bdcf75b0cd18a5651819ffdVesicle Tubulation with Self-Assembling DNA NanospringsGrome, Michael W.; Zhang, Zhao; Pincet, Frederic; Lin, ChenxiangAngewandte Chemie, International Edition (2018), 57 (19), 5330-5334CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A major goal of nanotechnol. and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane-deforming proteins, the authors designed DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coated membrane tubules emerge from spherical vesicles when DNA-origami polymn. or high membrane-surface coverage occurs. Unlike many previous methods, the DNA self-assembly-mediated membrane tubulation eliminates the need for detergents or top-down manipulation. The DNA-origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphol., underscoring the intricate interplay between lipid bilayers and vesicle-deforming DNA structures.
- 24Wang, T.; Schiffels, D.; Cuesta, S. M.; Fygenson, D. K.; Seeman, N. C. Design and Characterization of 1d Nanotubes and 2d Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles. J. Am. Chem. Soc. 2012, 134, 1606– 1616, DOI: 10.1021/ja207976qGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1OjtLfK&md5=23c775a8b633d109538c3411bd5e6b8dDesign and Characterization of 1D Nanotubes and 2D Periodic Arrays Self-Assembled from DNA Multi-Helix BundlesWang, Tong; Schiffels, Daniel; Martinez Cuesta, Sergio; Kuchnir Fygenson, Deborah; Seeman, Nadrian C.Journal of the American Chemical Society (2012), 134 (3), 1606-1616CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Among the key goals of structural DNA nanotechnol. are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helixes in the same plane (6HB+2), and the 6-helix bundle flanked by three helixes in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnol., so it is important to characterize their phys. properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 μm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.
- 25Schiffels, D.; Liedl, T.; Fygenson, D. K. Nanoscale Structure and Microscale Stiffness of DNA Nanotubes. ACS Nano 2013, 7, 6700– 6710, DOI: 10.1021/nn401362pGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFCrtL%252FI&md5=a7d952e66b12e5d6820977790ef88e3fNanoscale Structure and Microscale Stiffness of DNA NanotubesSchiffels, Daniel; Liedl, Tim; Fygenson, Deborah K.ACS Nano (2013), 7 (8), 6700-6710CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)We measure the stiffness of tiled DNA nanotubes (HX-tubes) as a function of their (defined) circumference by analyzing their micrometer-scale thermal deformations using fluorescence microscopy. We derive a model that relates nanoscale features of HX-tube architecture to the measured persistence lengths. Given the known stiffness of double-stranded DNA, we use this model to constrain the av. spacing between and effective stiffness of individual DNA duplexes in the tube. A key structural feature of tiled nanotubes that can affect stiffness is their potential to form with discrete amts. of twist of the DNA duplexes about the tube axis (supertwist). We visualize the supertwist of HX-tubes using electron microscopy of gold nanoparticles, attached to specific sites along the nanotube. This method reveals that HX-tubes tend not to form with supertwist unless forced by sequence design, and, even when forced, supertwist is reduced by elastic deformations of the underlying DNA lattice. We compare the hybridization energy gained upon closing a duplex sheet into a tube with the elastic energy paid for deforming the sheet to allow closure. In estg. the elastic energy we account for bending and twisting of the individual duplexes as well as shearing between them. We find the min. supertwist state has min. free energy, and global untwisting of forced supertwist is energetically favorable, consistent with our exptl. data. Finally, we show that attachment of Cy3 dyes or changing counterions can cause nanotubes to adopt a permanent writhe with micrometer-scale pitch and amplitude. We propose that the coupling of local twist and global counter-twist may be useful in characterizing perturbations of DNA structure.
- 26Kauert, D. J.; Kurth, T.; Liedl, T.; Seidel, R. Direct Mechanical Measurements Reveal the Material Properties of Three-Dimensional DNA Origami. Nano Lett. 2011, 11, 5558– 5563, DOI: 10.1021/nl203503sGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2ktLzM&md5=584b20313641a5b39ad32663bc2b787dDirect Mechanical Measurements Reveal the Material Properties of Three-Dimensional DNA OrigamiKauert, Dominik J.; Kurth, Thomas; Liedl, Tim; Seidel, RalfNano Letters (2011), 11 (12), 5558-5563CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The application of three-dimensional DNA origami objects as rigid mech. mediators or force sensing elements requires detailed knowledge about their complex mech. properties. Using magnetic tweezers, the authors directly measure the bending and torsional rigidities of four- and six-helix bundles assembled by this technique. Compared to duplex DNA, the authors find the bending rigidities to be greatly increased while the torsional rigidities are only moderately augmented. The authors present a mech. model explicitly including the crossovers between the individual helixes in the origami structure that reproduces the exptl. obsd. behavior. The authors' results provide an important basis for the future application of 3D DNA origami in nanomechanics.
- 27Castro, C. E.; Su, H. J.; Marras, A. E.; Zhou, L.; Johnson, J. Mechanical Design of DNA Nanostructures. Nanoscale 2015, 7, 5913– 5921, DOI: 10.1039/C4NR07153KGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitlamsrc%253D&md5=eb12536fddc1a44a0e7fb7a44d38fcecMechanical design of DNA nanostructuresCastro, Carlos E.; Su, Hai-Jun; Marras, Alexander E.; Zhou, Lifeng; Johnson, JoshuaNanoscale (2015), 7 (14), 5913-5921CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. Structural DNA nanotechnol. is a rapidly emerging field that has demonstrated great potential for applications such as single mol. sensing, drug delivery, and templating mol. components. As the applications of DNA nanotechnol. expand, a consideration of their mech. behavior is becoming essential to understand how these structures will respond to phys. interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mech. properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mech. stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mech. active nanomachines that can generate, transmit, and respond to phys. cues in mol. systems.
- 28Buchkovich, N. J.; Henne, W. M.; Tang, S.; Emr, S. D. Essential N-Terminal Insertion Motif Anchors the Escrt-Iii Filament During Mvb Vesicle Formation. Dev. Cell 2013, 27, 201– 214, DOI: 10.1016/j.devcel.2013.09.009Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1CltrvM&md5=677ff08a5c363db1f11c8936e186e52dEssential N-Terminal Insertion Motif Anchors the ESCRT-III Filament during MVB Vesicle FormationBuchkovich, Nicholas J.; Henne, William Mike; Tang, Shaogeng; Emr, Scott D.Developmental Cell (2013), 27 (2), 201-214CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)The endosomal sorting complexes required for transport (ESCRTs) have emerged as key cellular machinery that drive topol. unique membrane deformation and scission. Understanding how the ESCRT-III polymer interacts with membrane, promoting and/or stabilizing membrane deformation, is an important step in elucidating this sculpting mechanism. Using a combination of genetic and biochem. approaches, both in vivo and in vitro, we identify two essential modules required for ESCRT-III-membrane assocn.: an electrostatic cluster and an N-terminal insertion motif. Mutating either module in yeast causes cargo sorting defects in the MVB pathway. We show that the essential N-terminal insertion motif provides a stable anchor for the ESCRT-III polymer. By replacing this N-terminal motif with well-characterized membrane insertion modules, we demonstrate that the N terminus of the Snf7 subunit has been tuned to maintain the topol. constraints assocd. with ESCRT-III-filament-mediated membrane invagination and vesicle formation. Our results provide insights into the spatially unique, ESCRT-III-mediated membrane remodeling.
- 29Kim, D. N.; Kilchherr, F.; Dietz, H.; Bathe, M. Quantitative Prediction of 3d Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res. 2012, 40, 2862– 2868, DOI: 10.1093/nar/gkr1173Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xls1Snu7k%253D&md5=5570389892b346fe6198edd9361e21e1Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructuresKim, Do-Nyun; Kilchherr, Fabian; Dietz, Hendrik; Bathe, MarkNucleic Acids Research (2012), 40 (7), 2862-2868CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA nanotechnol. enables the programmed synthesis of intricate nanometer-scale structures for diverse applications in materials and biol. science. Precise control over the 3D soln. shape and mech. flexibility of target designs is important to achieve desired functionality. Because exptl. validation of designed nanostructures is time-consuming and cost-intensive, predictive phys. models of nanostructure shape and flexibility have the capacity to enhance dramatically the design process. Here, the authors significantly extend and exptl. validate a computational modeling framework for DNA origami previously presented as CanDo A primer to scaffolded DNA origami. 3D soln. shape and flexibility are predicted from basepair connectivity maps now accounting for nicks in the DNA double helix, entropic elasticity of single-stranded DNA, and distant crossovers required to model wireframe structures, in addn. to previous modeling that accounted only for the canonical twist, bend and stretch stiffness of double-helical DNA domains. Systematic exptl. validation of nanostructure flexibility mediated by internal crossover d. probed using a 32-helix DNA bundle demonstrates for the first time that our model not only predicts the 3D soln. shape of complex DNA nanostructures but also their mech. flexibility. Thus, our model represents an important advance in the quant. understanding of DNA-based nanostructure shape and flexibility, and we anticipate that this model will increase significantly the no. and variety of synthetic nanostructures designed using nucleic acids.
- 30Myhrvold, C.; Baym, M.; Hanikel, N.; Ong, L. L.; Gootenberg, J. S.; Yin, P. Barcode Extension for Analysis and Reconstruction of Structures. Nat. Commun. 2017, 8, 14698, DOI: 10.1038/ncomms14698Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkslOjsLg%253D&md5=9bcd74283733debc855798ac30d4fceaBarcode extension for analysis and reconstruction of structuresMyhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L.; Gootenberg, Jonathan S.; Yin, PengNature Communications (2017), 8 (), 14698CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously det. the presence of individual, labeled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quant. measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.
- 31Benson, E.; Mohammed, A.; Rayneau-Kirkhope, D.; Gadin, A.; Orponen, P.; Hogberg, B. Effects of Design Choices on the Stiffness of Wireframe DNA Origami Structures. ACS Nano 2018, 12, 9291– 9299, DOI: 10.1021/acsnano.8b04148Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1Cns7nF&md5=edbea1de7199f7ac57283421cad4fedaEffects of Design Choices on the Stiffness of Wireframe DNA Origami StructuresBenson, Erik; Mohammed, Abdulmelik; Rayneau-Kirkhope, Daniel; Gaadin, Andreas; Orponen, Pekka; Hoegberg, BjoernACS Nano (2018), 12 (9), 9291-9299CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)DNA origami is a powerful method for the creation of 3D nanoscale objects, and in the past few years, interest in wireframe origami designs has increased due to their potential for biomedical applications. In DNA wireframe designs, the construction material is double-stranded DNA, which has a persistence length of around 50 nm. In this work, the authors study the effect of various design choices on the stiffness vs. final size of nanoscale wireframe rods, given the constraints on origami designs set by the DNA origami scaffold size. An initial theor. anal. predicts two competing mechanisms limiting rod stiffness, whose balancing results in an optimal edge length. For small edge lengths, the bending of the rod's overall frame geometry is the dominant factor, while the flexibility of individual DNA edges has a greater contribution at larger edge lengths. The authors evaluate the design choices through simulations and expts. and find that the stiffness of the structures increases with the no. of sides in the cross-section polygon and that there are indications of an optimal member edge length. The authors also ascertain the effect of nicked DNA edges on the stiffness of the wireframe rods and demonstrate that ligation of the staple breakpoint nicks reduces the obsd. flexibility. The simulations also indicate that the persistence length of wireframe DNA structures significantly decreases with increasing monovalent salt concn.
- 32Khmelinskaia, 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, DOI: 10.1088/0022-3727/49/19/194001Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWrsb%252FK&md5=ea2abc7e490dd9b694d008f613daba67Effect of anchor positioning on binding and diffusion of elongated 3D DNA nanostructures on lipid membranesKhmelinskaia, Alena; Franquelim, Henri G.; Petrov, Eugene P.; Schwille, PetraJournal of Physics D: Applied Physics (2016), 49 (19), 194001/1-194001/11CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)DNA origami is a state-of-the-art technol. that enables the fabrication of nano-objects with defined shapes, to which functional moieties, such as lipophilic anchors, can be attached with a nanometer scale precision. Although binding of DNA origami to lipid membranes has been extensively demonstrated, the specific requirements necessary for membrane attachment are greatly overlooked. Here, we designed a set of amphipathic rectangular-shaped DNA origami structures with varying placement and no. of chol-TEG anchors used for membrane attachment. Single- and multiple-cholesteryl-modified origami nanostructures were produced and studied in terms of their membrane localization, d. and dynamics. We show that the positioning of at least two chol-TEG moieties near the corners is essential to ensure efficient membrane binding of large DNA nanostructures. Quant. fluorescence correlation spectroscopy data further confirm that increasing the no. of corner-positioned chol-TEG anchors lowers the dynamics of flat DNA origami structures on freestanding membranes. Taken together, our approach provides the first evidence of the importance of the location in addn. to the no. of hydrophobic moieties when rationally designing minimal DNA nanostructures with controlled membrane binding.
- 33Khmelinskaia, A.; Mucksch, J.; Petrov, E. P.; Franquelim, H. G.; Schwille, P. Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers. Langmuir 2018, 34, 14921– 14931, DOI: 10.1021/acs.langmuir.8b01850Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOqsrjE&md5=1df97032f81594bc11ff6ddb37d329f6Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA SpacersKhmelinskaia, Alena; Muecksch, Jonas; Petrov, Eugene P.; Franquelim, Henri G.; Schwille, PetraLangmuir (2018), 34 (49), 14921-14931CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)DNA origami nanotechnol. is increasingly used to mimic membrane-assocd. biophys. phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, the authors used a set of elongated DNA origami structures with varying placement and no. of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. The results show that the location and no. of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct phys. properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, the authors provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.
- 34Nguyen, N.; Shteyn, V.; Melia, T. J. Sensing Membrane Curvature in Macroautophagy. J. Mol. Biol. 2017, 429, 457– 472, DOI: 10.1016/j.jmb.2017.01.006Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCgtLc%253D&md5=69b3124b6fbf60a64a84273150b3eda0Sensing membrane curvature in macroautophagyNguyen, Nathan; Shteyn, Vladimir; Melia, Thomas J.Journal of Molecular Biology (2017), 429 (4), 457-472CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)A review. In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo formation of a new organelle called the autophagosome, which either surrounds and sequesters random portions of the cytoplasm or selectively targets individual intracellular challenges. Thus, the autophagosome is a flexible membrane platform with dimensions that ultimately depend upon the target cargo. The intermediate membrane, termed the phagophore or isolation membrane, is a cup-like structure with a clear concave face and a highly curved rim. The phagophore is largely devoid of integral membrane proteins; thus, its shape and size are governed by peripherally assocd. membrane proteins and possibly by the lipid compn. of the membrane itself. Growth along the phagophore rim marks the progress of both organelle expansion and ultimately organelle closure around a particular cargo. These 2 properties, a reliance on peripheral membrane proteins and a structurally distinct membrane architecture, suggest that the ability to target or manipulate membrane curvature might be an essential activity of proteins functioning in this pathway. Here, the authors discuss the extent to which membranes are naturally curved at each of the cellular sites believed to engage in autophagosome formation, review basic mechanisms used to sense this curvature, and then summarize the existing literature concerning which autophagy proteins are capable of curvature recognition.
- 35Bhatia, V. K.; Hatzakis, N. S.; Stamou, D. A Unifying Mechanism Accounts for Sensing of Membrane Curvature by Bar Domains, Amphipathic Helices and Membrane-Anchored Proteins. Semin. Cell Dev. Biol. 2010, 21, 381– 390, DOI: 10.1016/j.semcdb.2009.12.004Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkvFKrt7o%253D&md5=d25e138be5fca2b7a8a235c938694f4cA unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteinsBhatia, Vikram Kjoeller; Hatzakis, Nikos S.; Stamou, DimitriosSeminars in Cell & Developmental Biology (2010), 21 (4), 381-390CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)A review. The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein compn. Here we review recent data on membrane curvature sensing by three structurally unrelated motifs: BAR domains, amphipathic helixes and membrane-anchored proteins. We discuss the conclusion that the curvature of the BAR dimer is not responsible for sensing and that the sensing properties of all three motifs can be rationalized by the physicochem. properties of the curved membrane itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biol.
- 36Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano. Nucleic Acids Res. 2009, 37, 5001– 5006, DOI: 10.1093/nar/gkp436Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntbzE&md5=aa99732c1666373a70e9b7b4de6e6d5dRapid prototyping of 3D DNA-origami shapes with caDNAnoDouglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.
- 37Bellot, G.; McClintock, M. A.; Chou, J. J.; Shih, W. M. DNA Nanotubes for Nmr Structure Determination of Membrane Proteins. Nat. Protoc. 2013, 8, 755– 770, DOI: 10.1038/nprot.2013.037Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmtlShsbo%253D&md5=21847aedab3e0bcecdca6028ac8c92deDNA nanotubes for NMR structure determination of membrane proteinsBellot, Gaetan; McClintock, Mark A.; Chou, James J.; Shih, William M.Nature Protocols (2013), 8 (4), 755-770, 16 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Finding a way to det. the structures of integral membrane proteins using soln. NMR (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dil. liq. crystals for application as weak-alignment media in soln. NMR structure detn. of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable pos. charge as well as modulation of mol. alignment, toward collection of linearly independent restraints, can be introduced by reducing the neg. charge of DNA nanotubes using counter ions and small DNA-binding mols. This detergent-resistant liq.-crystal medium offers a no. of properties conducive for membrane protein alignment, including high-yield prodn., thermal stability, buffer compatibility and structural programmability. Prodn. of sufficient nanotubes for four or five NMR expts. can be completed in 1 wk by a single individual.
- 38Lin, C.; Perrault, S. D.; Kwak, M.; Graf, F.; Shih, W. M. Purification of DNA-Origami Nanostructures by Rate-Zonal Centrifugation. Nucleic Acids Res. 2013, 41, e40, DOI: 10.1093/nar/gks1070Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFyjurg%253D&md5=55de3fad844f9e5e0802d5abca92f5afPurification of DNA-origami nanostructures by rate-zonal centrifugationLin, Chenxiang; Perrault, Steven D.; Kwak, Minseok; Graf, Franziska; Shih, William M.Nucleic Acids Research (2013), 41 (2), e40CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Most previously reported methods for purifying DNA-origami nanostructures have relied on agarose-gel electrophoresis (AGE) for sepn. Although AGE is routinely used to yield 0.1-1 μg purified DNA nanostructures, obtaining >100 μg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extn., desalting, and concn. steps. Here, the authors present a readily scalable purifn. approach utilizing rate-zonal centrifugation, which provides comparable sepn. resoln. as AGE. The DNA nanostructures remain in aq. soln. throughout the purifn. process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA-intercalating dyes. Seven distinct 3-dimensional DNA-origami constructs were purified at the scale of 0.1-100 μg (final yield) per centrifuge tube, showing the versatility of this method. Given the com. available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for prepn. of larger amts. (e.g., milligram quantities) of DNA nanostructures.
- 39Stahl, E.; Martin, T. G.; Praetorius, F.; Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew. Chem., Int. Ed. 2014, 53, 12735– 12740, DOI: 10.1002/anie.201405991Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvV2mtrrM&md5=5d749b225ce5405de5c89eeb1a5b1897Facile and Scalable Preparation of Pure and Dense DNA Origami SolutionsStahl, Evi; Martin, Thomas G.; Praetorius, Florian; Dietz, HendrikAngewandte Chemie, International Edition (2014), 53 (47), 12735-12740CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA has become a prime material for assembling complex three-dimensional objects that promise utility in various areas of application. However, achieving user-defined goals with DNA objects has been hampered by the difficulty to prep. them at arbitrary concns. and in user-defined soln. conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)-induced depletion of species with high mol. wt. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient sepn. from non-integrated DNA strands. DNA objects may be prepd. at concns. up to the limit of soly., including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self-assembled DNA objects.
- 40Lamour, G.; Kirkegaard, J. B.; Li, H.; Knowles, T. P.; Gsponer, J. Easyworm: An Open-Source Software Tool to Determine the Mechanical Properties of Worm-Like Chains. Source Code Biol. Med. 2014, 9, 16, DOI: 10.1186/1751-0473-9-16Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cbptVOhtQ%253D%253D&md5=6462166224a41bc00fd68752c3cc6a63Easyworm: an open-source software tool to determine the mechanical properties of worm-like chainsLamour Guillaume; Kirkegaard Julius B; Knowles Tuomas Pj; Li Hongbin; Gsponer JorgSource code for biology and medicine (2014), 9 (), 16 ISSN:1751-0473.BACKGROUND: A growing spectrum of applications for natural and synthetic polymers, whether in industry or for biomedical research, demands for fast and universally applicable tools to determine the mechanical properties of very diverse polymers. To date, determining these properties is the privilege of a limited circle of biophysicists and engineers with appropriate technical skills. FINDINGS: Easyworm is a user-friendly software suite coded in MATLAB that simplifies the image analysis of individual polymeric chains and the extraction of the mechanical properties of these chains. Easyworm contains a comprehensive set of tools that, amongst others, allow the persistence length of single chains and the Young's modulus of elasticity to be calculated in multiple ways from images of polymers obtained by a variety of techniques (e.g. atomic force microscopy, electron, contrast-phase, or epifluorescence microscopy). CONCLUSIONS: Easyworm thus provides a simple and efficient tool for specialists and non-specialists alike to solve a common problem in (bio)polymer science. Stand-alone executables and shell scripts are provided along with source code for further development.
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Abstract
Figure 1
Figure 1. Designs and assembly of DNA-origami nanosprings of varying thicknesses. (a) Cartoon models depicting the 24, 12, and 6 helix-bundle (HB) DNA curls and their linker-triggered polymerization into nanosprings. Each DNA double-helix and DNA-conjugated amphipathic peptide (16 per monomer) are represented by a curved gray rod and a purple sphere, respectively. Negative-stain TEM images of peptide-labeled DNA curls and nanosprings are located next to the corresponding schematics. Scale bars = 100 nm. (b) Top views of DNA-curl models (top) and schematic drawings of the Cy5-modified DNA-peptide conjugate that decorate the interiors of the DNA curls (bottom). (c) An agarose gel resolving DNA curls with varying numbers of inward-facing ssDNA handles (Handle #) in the presence and absence of Cy5-modified antihandle-peptide (AH-peptide) conjugates. Bands that contain monomeric DNA curls and misfolded aggregates are boxed and labeled with asterisks (*) and daggers (†), respectively. Pseudocolor fluorescence: red = Cy5, green = ethidium bromide.
Figure 2
Figure 2. Large unilamellar vesicle (LUV) binding and tubulation activities of self-assembling DNA curls of varying thicknesses and membrane anchor densities. (a) Schematic illustration of the experiment procedures used to test DNA-curl induced LUV tubulation with and without linkers. (b) Fractionated density gradients containing Cy5-labeled DNA curls with 16 or 0 amphipathic peptides per monomer (Pep #) and rhodamine-labeled LUVs after isopycnic centrifugation. Pseudocolor fluorescence of recovered fractions: red = Cy5, blue = rhodamine. Smaller numbers denote lighter fractions. (c) Normalized abundances of lipid tubules generated by 24, 12, or 6 helix-bundle (HB) curls with 16 or 7 peptides per monomer in the presence or absence of linker. For each measurement, 750–2000 vesicles were surveyed. (d) Representative negative-stain TEM images of LUVs tubulated by DNA curls (16 peptide per monomer) of different thicknesses, with and without DNA linkers. Scale bars = 400 nm.
Figure 3
Figure 3. Impact of DNA-nanospring thickness on lipid-tubule morphologies. (a) TEM images of lipid tubules covered by 24, 12, and 6 helix-bundle (HB) nanosprings. Scale bars = 100 nm. (b) Average tubule widths as a result of polymerizing 24, 12, and 6HB DNA curls (16 amphipathic peptides per monomer) mixed with LUVs. Means are weighted for the tubule lengths. Error bars show standard deviations. (c) Scatter plots showing the widths of lipid tubules versus the apparent pitches of the tubule-associating 24 and 12HB nanosprings (defined as the length of nanospring-coated tubule divided by the cumulative helical turns of DNA filaments).
Figure 4
Figure 4. Cartoon models and the corresponding TEM images contrasting the compressed 24 and 12 helix-bundle (HB) DNA nanosprings (gray, blue, and red) intertwining around lipid tubules (tan). The measured apparent and actual pitches of nanosprings are labeled at the bottom and top of each model, respectively. The “see-through” box reveals the different surface densities of peptides (purple dots) introduced by the 24 and 12HB nanosprings. Scale bar = 100 nm.
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- 5Frost, A.; Unger, V. M.; De Camilli, P. The Bar Domain Superfamily: Membrane-Molding Macromolecules. Cell 2009, 137, 191– 196, DOI: 10.1016/j.cell.2009.04.010Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmvVaiurg%253D&md5=c2355b3dbd92094526b075074f376e88The BAR domain superfamily: membrane-molding macromoleculesFrost, Adam; Unger, Vinzenz M.; De Camilli, PietroCell (Cambridge, MA, United States) (2009), 137 (2), 191-196CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.
- 6Hurley, J. H.; Hanson, P. I. Membrane Budding and Scission by the Escrt Machinery: It’s All in the Neck. Nat. Rev. Mol. Cell Biol. 2010, 11, 556– 566, DOI: 10.1038/nrm2937Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXotFeltL0%253D&md5=9115205a75cdc132eb475f09871fb308Membrane budding and scission by the ESCRT machinery: it's all in the neckHurley, James H.; Hanson, Phyllis I.Nature Reviews Molecular Cell Biology (2010), 11 (8), 556-566CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. The endosomal sorting complexes required for transport (ESCRTs) catalyze one of the most unusual membrane remodeling events in cell biol. ESCRT-I and ESCRT-II direct membrane budding away from the cytosol by stabilizing bud necks without coating the buds and without being consumed in the buds. ESCRT-III cleaves the bud necks from their cytosolic faces. ESCRT-III-mediated membrane neck cleavage is crucial for many processes, including the biogenesis of multivesicular bodies, viral budding, cytokinesis, and, probably, autophagy. Recent studies of ultrastructures induced by ESCRT-III overexpression in cells and the in vitro reconstitution of the budding and scission reactions have led to breakthroughs in understanding these remarkable membrane reactions.
- 7Ford, M. G.; Mills, I. G.; Peter, B. J.; Vallis, Y.; Praefcke, G. J.; Evans, P. R.; McMahon, H. T. Curvature of Clathrin-Coated Pits Driven by Epsin. Nature 2002, 419, 361– 366, DOI: 10.1038/nature01020Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XntlGgsLc%253D&md5=c4874e53217ae4239c2809e5471c5279Curvature of clathrin-coated pits driven by epsinFord, Marijn G. J.; Mills, Ian G.; Peter, Brian J.; Vallis, Yvonne; Praefcke, Gerrit J. K.; Evans, Philip R.; McMahon, Harvey T.Nature (London, United Kingdom) (2002), 419 (6905), 361-366CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P2 in conjunction with clathrin polymn. We have discovered that the formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.
- 8Roux, A.; Cappello, G.; Cartaud, J.; Prost, J.; Goud, B.; Bassereau, P. A Minimal System Allowing Tubulation with Molecular Motors Pulling on Giant Liposomes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5394– 5399, DOI: 10.1073/pnas.082107299Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjtFKlsLs%253D&md5=c43c4d4592dc899963d857aae7f22ad8A minimal system allowing tubulation with molecular motors pulling on giant liposomesRoux, Aurelien; Cappello, Giovanni; Cartaud, Jean; Prost, Jacques; Goud, Bruno; Bassereau, PatriciaProceedings of the National Academy of Sciences of the United States of America (2002), 99 (8), 5394-5399CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The elucidation of phys. and mol. mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that mol. motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin mols. have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was obsd. when kinesins were directly bound to the GUV membrane, suggesting that it is crit. to distribute the load on both lipids and motors by means of beads. A kinetic anal. shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled expts. aimed at the understanding of basic mechanisms in intracellular transport.
- 9Takei, K.; Slepnev, V. I.; Haucke, V.; De Camilli, P. Functional Partnership between Amphiphysin and Dynamin in Clathrin-Mediated Endocytosis. Nat. Cell Biol. 1999, 1, 33– 39, DOI: 10.1038/9004Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXksVKitrw%253D&md5=f66e85a3d5fb1a392d3642d5e1520139Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosisTakei, Kohji; Slepnev, Vladimir I.; Haucke, Volker; De Camilli, PietroNature Cell Biology (1999), 1 (1), 33-39CODEN: NCBIFN; ISSN:1465-7392. (Macmillan Magazines Ltd)Amphiphysin, a protein that is highly concd. in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphol. evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.
- 10Seeman, N. C. Nucleic Acid Junctions and Lattices. J. Theor. Biol. 1982, 99, 237– 247, DOI: 10.1016/0022-5193(82)90002-9Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3sXhtlWhuw%253D%253D&md5=82c15bbb6dadb9b373d9da1a68ba23e3Nucleic acid junctions and latticesSeeman, Nadrian C.Journal of Theoretical Biology (1982), 99 (2), 237-47CODEN: JTBIAP; ISSN:0022-5193.It is possible to generate sequences of oligomeric nucleic acids which will preferentially assoc. to form migrationally immobile junctions, rather than linear duplexes. These structures are predicted on the maximization of Watson-Crick base pairing and the lack of sequence symmetry customarily found in their analogs in living systems. Criteria which oligonucleotide sequences must fulfill to yield these junction structures are presented. The generable junctions are nexuses, from which 3-8 double helices may emanate. Each junction may be treated as a macromol. valence cluster, and individual clusters may be linked together directly, or with pieces of linear DNA interspersed between them. This covalent linkage can be done with enormous specificity, using sticky-ended ligation techniques. It appears to be possible to generate covalently joined 3-dimensional networks of nucleic acids which are periodic in connectivity and perhaps in space.
- 11Rothemund, P. W. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297– 302, DOI: 10.1038/nature04586Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XitlKgu7g%253D&md5=583caefdda9b1deb5d3f2ef78d9e6ecbFolding DNA to create nanoscale shapes and patternsRothemund, Paul W. K.Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
- 12Douglas, 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, DOI: 10.1038/nature08016Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtFKmtbs%253D&md5=9c6876c4b0358a9f31a0f4aaa5ba855aSelf-assembly of DNA into nanoscale three-dimensional shapesDouglas, Shawn M.; Dietz, Hendrik; Liedl, Tim; Hogberg, Bjorn; Graf, Franziska; Shih, William M.Nature (London, United Kingdom) (2009), 459 (7245), 414-418CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Mol. self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase scaffold strand' that is folded into a flat array of antiparallel helixes by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helixes constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concns. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manuf. of sophisticated devices bearing features on the nanometer scale.
- 13Dietz, H.; Douglas, S. M.; Shih, W. M. Folding DNA into Twisted and Curved Nanoscale Shapes. Science 2009, 325, 725– 730, DOI: 10.1126/science.1174251Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptl2guro%253D&md5=8dd007a0fba43e591d45b3640b129f10Folding DNA into Twisted and Curved Nanoscale ShapesDietz, Hendrik; Douglas, Shawn M.; Shih, William M.Science (Washington, DC, United States) (2009), 325 (5941), 725-730CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)We demonstrate the ability to engineer complex shapes that twist and curve at the nanoscale from DNA. Through programmable self-assembly, strands of DNA are directed to form a custom-shaped bundle of tightly crosslinked double helixes, arrayed in parallel to their helical axes. Targeted insertions and deletions of base pairs cause the DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quant. controlled, and a radius of curvature as tight as 6 nm was achieved. We also combined multiple curved elements to build several different types of intricate nanostructures, such as a wireframe beach ball or square-toothed gears.
- 14Seeman, N. C.; Sleiman, H. F. DNA Nanotechnology. Nat. Rev. Mater. 2018, 3, 17068, DOI: 10.1038/natrevmats.2017.68Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslOntLbM&md5=a8683cd5a65e013464f37bb6383853b4DNA nanotechnologySeeman, Nadrian C.; Sleiman, Hanadi F.Nature Reviews Materials (2018), 3 (1), 17068CODEN: NRMADL; ISSN:2058-8437. (Nature Research)DNA is the mol. that stores and transmits genetic information in biol. systems. The field of DNA nanotechnol. takes this mol. out of its biol. context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnol., and has been revolutionary in our ability to control mol. self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomol. structure detn., drug delivery and synthetic biol. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.
- 15Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584– 12640, DOI: 10.1021/acs.chemrev.6b00825Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1CjsL0%253D&md5=db637726b0112f270fe979339d24f96aDNA Origami: Scaffolds for Creating Higher Order StructuresHong, Fan; Zhang, Fei; Liu, Yan; Yan, HaoChemical Reviews (Washington, DC, United States) (2017), 117 (20), 12584-12640CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. DNA has become one of the most extensively used mol. building blocks for engineering self-assembling materials. DNA origami is a technique that uses hundreds of short DNA oligonucleotides, called staple strands, to fold a long single-stranded DNA, which is called a scaffold strand, into various designer nanoscale architectures. DNA origami has dramatically improved the complexity and scalability of DNA nanostructures. Due to its high degree of customization and spatial addressability, DNA origami provides a versatile platform with which to engineer nanoscale structures and devices that can sense, compute, and actuate. These capabilities open up opportunities for a broad range of applications in chem., biol., physics, material science, and computer science that have often required programmed spatial control of mols. and atoms in three-dimensional (3D) space. This review provides a comprehensive survey of recent developments in DNA origami structure, design, assembly, and directed self-assembly, as well as its broad applications.
- 16Zhang, Z.; Yang, Y.; Pincet, F.; Llaguno, M. C.; Lin, C. Placing and Shaping Liposomes with Reconfigurable DNA Nanocages. Nat. Chem. 2017, 9, 653– 659, DOI: 10.1038/nchem.2802Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCrtb3L&md5=e5e94cb3f83dba3ca54caa3428bcce4cPlacing and shaping liposomes with reconfigurable DNA nanocagesZhang, Zhao; Yang, Yang; Pincet, Frederic; Llaguno, Marc C.; Lin, ChenxiangNature Chemistry (2017), 9 (7), 653-659CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The diverse structure and regulated deformation of lipid bilayer membranes are among a cell's most fascinating features. Artificial membrane-bound vesicles, known as liposomes, are versatile tools for modeling biol. membranes and delivering foreign objects to cells. To fully mimic the complexity of cell membranes and optimize the efficiency of delivery vesicles, controlling liposome shape (both statically and dynamically) is of utmost importance. Here, we report the assembly, arrangement and remodeling of liposomes with designer geometry: all of which are exquisitely controlled by a set of modular, reconfigurable DNA nanocages. Tubular and toroid shapes, among others, are transcribed from DNA cages to liposomes with high fidelity, giving rise to membrane curvatures present in cells yet previously difficult to construct in vitro. Moreover, the conformational changes of DNA cages drive membrane fusion and bending with predictable outcomes, opening up opportunities for the systematic study of membrane mechanics.
- 17Czogalla, 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. 2015, 54, 6501– 6505, DOI: 10.1002/anie.201501173Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmslart78%253D&md5=cca84a507f151e29a2a5bf80059c6126Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane VesiclesCzogalla, Aleksander; Kauert, Dominik J.; Franquelim, Henri G.; Uzunova, Veselina; Zhang, Yixin; Seidel, Ralf; Schwille, PetraAngewandte Chemie, International Edition (2015), 54 (22), 6501-6505CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report a synthetic biol.-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions giving ordered arrays on the membrane. Such a tight and regular arrangement makes the authors' DNA origami capable of deforming free-standing lipid membranes, mimicking the biol. activity of coat-forming proteins, for example, from the I-/F-BAR family.
- 18Kocabey, 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, DOI: 10.1021/acsnano.5b00161Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjslemsbc%253D&md5=1599e15c303bf8f52be09c43cf8f4a3cMembrane-Assisted Growth of DNA Origami Nanostructure ArraysKocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M.; Simmel, Friedrich C.; Liedl, TimACS Nano (2015), 9 (4), 3530-3539CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Biol. membranes fulfill many important tasks within living organisms. In addn. to sepg. cellular vols., membranes confine the space available to membrane-assocd. proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. The authors here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors, a three-layered rectangular block and a Y-shaped DNA structure, to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coeffs. depending on their size and no. of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures assocd. into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. The authors' results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.
- 19Howorka, S. Nanotechnology. Changing of the Guard. Science 2016, 352, 890– 891, DOI: 10.1126/science.aaf5154Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVSrs7c%253D&md5=cd1bbdcddd6afb5666d0e21d7713bd51Changing of the guardHoworka, StefanScience (Washington, DC, United States) (2016), 352 (6288), 890-891CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)There is no expanded citation for this reference.
- 20Birkholz, O.; Burns, J. R.; Richter, C. P.; Psathaki, O. E.; Howorka, S.; Piehler, J. Multi-Functional DNA Nanostructures That Puncture and Remodel Lipid Membranes into Hybrid Materials. Nat. Commun. 2018, 9, 1521, DOI: 10.1038/s41467-018-02905-wGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjktFKqtg%253D%253D&md5=8f8cbce2240bfd769f91e2bf3900f0f8Multi-functional DNA nanostructures that puncture and remodel lipid membranes into hybrid materialsBirkholz Oliver; Richter Christian P; Psathaki Olympia E; Piehler Jacob; Burns Jonathan R; Howorka StefanNature communications (2018), 9 (1), 1521 ISSN:.Synthetically replicating key biological processes requires the ability to puncture lipid bilayer membranes and to remodel their shape. Recently developed artificial DNA nanopores are one possible synthetic route due to their ease of fabrication. However, an unresolved fundamental question is how DNA nanopores bind to and dynamically interact with lipid bilayers. Here we use single-molecule fluorescence microscopy to establish that DNA nanopores carrying cholesterol anchors insert via a two-step mechanism into membranes. Nanopores are furthermore shown to locally cluster and remodel membranes into nanoscale protrusions. Most strikingly, the DNA pores can function as cytoskeletal components by stabilizing autonomously formed lipid nanotubes. The combination of membrane puncturing and remodeling activity can be attributed to the DNA pores' tunable transition between two orientations to either span or co-align with the lipid bilayer. This insight is expected to catalyze the development of future functional nanodevices relevant in synthetic biology and nanobiotechnology.
- 21Gopfrich, K.; Zettl, T.; Meijering, A. E.; Hernandez-Ainsa, S.; Kocabey, S.; Liedl, T.; Keyser, U. F. DNA-Tile Structures Induce Ionic Currents through Lipid Membranes. Nano Lett. 2015, 15, 3134– 3138, DOI: 10.1021/acs.nanolett.5b00189Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlsFaqtL8%253D&md5=88671fefb314500fadfcc611970480cbDNA-tile structures induce ionic currents through lipid membranesGopfrich, Kerstin; Zettl, Thomas; Meijering, Anna E. C.; Hernandez-Ainsa, Silvia; Kocabey, Samet; Liedl, Tim; Keyser, Ulrich F.Nano Letters (2015), 15 (5), 3134-3138CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diam. of 5 nm and a mol. wt. of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biol. ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biol. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.
- 22Franquelim, H. G.; Khmelinskaia, A.; Sobczak, J. P.; Dietz, H.; Schwille, P. Membrane Sculpting by Curved DNA Origami Scaffolds. Nat. Commun. 2018, 9, 811, DOI: 10.1038/s41467-018-03198-9Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MrkvVOlsw%253D%253D&md5=308b629abe07442332c291a889c7a404Membrane sculpting by curved DNA origami scaffoldsFranquelim Henri G; Khmelinskaia Alena; Schwille Petra; Khmelinskaia Alena; Sobczak Jean-Philippe; Dietz HendrikNature communications (2018), 9 (1), 811 ISSN:.Membrane sculpting and transformation is essential for many cellular functions, thus being largely regulated by self-assembling and self-organizing protein coats. Their functionality is often encoded by particular spatial structures. Prominent examples are BAR domain proteins, the 'banana-like' shapes of which are thought to aid scaffolding and membrane tubulation. To elucidate whether 3D structure can be uncoupled from other functional features of complex scaffolding proteins, we hereby develop curved DNA origami in various shapes and stacking features, following the presumable design features of BAR proteins, and characterize their ability for membrane binding and transformation. We show that dependent on curvature, membrane affinity and surface density, DNA origami coats can indeed reproduce the activity of membrane-sculpting proteins such as BAR, suggesting exciting perspectives for using them in bottom-up approaches towards minimal biomimetic cellular machineries.
- 23Grome, M. W.; Zhang, Z.; Pincet, F.; Lin, C. Vesicle Tubulation with Self-Assembling DNA Nanosprings. Angew. Chem., Int. Ed. 2018, 57, 5330– 5334, DOI: 10.1002/anie.201800141Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnt1elsL0%253D&md5=5da4fa7a1bdcf75b0cd18a5651819ffdVesicle Tubulation with Self-Assembling DNA NanospringsGrome, Michael W.; Zhang, Zhao; Pincet, Frederic; Lin, ChenxiangAngewandte Chemie, International Edition (2018), 57 (19), 5330-5334CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A major goal of nanotechnol. and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane-deforming proteins, the authors designed DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coated membrane tubules emerge from spherical vesicles when DNA-origami polymn. or high membrane-surface coverage occurs. Unlike many previous methods, the DNA self-assembly-mediated membrane tubulation eliminates the need for detergents or top-down manipulation. The DNA-origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphol., underscoring the intricate interplay between lipid bilayers and vesicle-deforming DNA structures.
- 24Wang, T.; Schiffels, D.; Cuesta, S. M.; Fygenson, D. K.; Seeman, N. C. Design and Characterization of 1d Nanotubes and 2d Periodic Arrays Self-Assembled from DNA Multi-Helix Bundles. J. Am. Chem. Soc. 2012, 134, 1606– 1616, DOI: 10.1021/ja207976qGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1OjtLfK&md5=23c775a8b633d109538c3411bd5e6b8dDesign and Characterization of 1D Nanotubes and 2D Periodic Arrays Self-Assembled from DNA Multi-Helix BundlesWang, Tong; Schiffels, Daniel; Martinez Cuesta, Sergio; Kuchnir Fygenson, Deborah; Seeman, Nadrian C.Journal of the American Chemical Society (2012), 134 (3), 1606-1616CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Among the key goals of structural DNA nanotechnol. are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helixes in the same plane (6HB+2), and the 6-helix bundle flanked by three helixes in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnol., so it is important to characterize their phys. properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 μm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.
- 25Schiffels, D.; Liedl, T.; Fygenson, D. K. Nanoscale Structure and Microscale Stiffness of DNA Nanotubes. ACS Nano 2013, 7, 6700– 6710, DOI: 10.1021/nn401362pGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFCrtL%252FI&md5=a7d952e66b12e5d6820977790ef88e3fNanoscale Structure and Microscale Stiffness of DNA NanotubesSchiffels, Daniel; Liedl, Tim; Fygenson, Deborah K.ACS Nano (2013), 7 (8), 6700-6710CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)We measure the stiffness of tiled DNA nanotubes (HX-tubes) as a function of their (defined) circumference by analyzing their micrometer-scale thermal deformations using fluorescence microscopy. We derive a model that relates nanoscale features of HX-tube architecture to the measured persistence lengths. Given the known stiffness of double-stranded DNA, we use this model to constrain the av. spacing between and effective stiffness of individual DNA duplexes in the tube. A key structural feature of tiled nanotubes that can affect stiffness is their potential to form with discrete amts. of twist of the DNA duplexes about the tube axis (supertwist). We visualize the supertwist of HX-tubes using electron microscopy of gold nanoparticles, attached to specific sites along the nanotube. This method reveals that HX-tubes tend not to form with supertwist unless forced by sequence design, and, even when forced, supertwist is reduced by elastic deformations of the underlying DNA lattice. We compare the hybridization energy gained upon closing a duplex sheet into a tube with the elastic energy paid for deforming the sheet to allow closure. In estg. the elastic energy we account for bending and twisting of the individual duplexes as well as shearing between them. We find the min. supertwist state has min. free energy, and global untwisting of forced supertwist is energetically favorable, consistent with our exptl. data. Finally, we show that attachment of Cy3 dyes or changing counterions can cause nanotubes to adopt a permanent writhe with micrometer-scale pitch and amplitude. We propose that the coupling of local twist and global counter-twist may be useful in characterizing perturbations of DNA structure.
- 26Kauert, D. J.; Kurth, T.; Liedl, T.; Seidel, R. Direct Mechanical Measurements Reveal the Material Properties of Three-Dimensional DNA Origami. Nano Lett. 2011, 11, 5558– 5563, DOI: 10.1021/nl203503sGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2ktLzM&md5=584b20313641a5b39ad32663bc2b787dDirect Mechanical Measurements Reveal the Material Properties of Three-Dimensional DNA OrigamiKauert, Dominik J.; Kurth, Thomas; Liedl, Tim; Seidel, RalfNano Letters (2011), 11 (12), 5558-5563CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)The application of three-dimensional DNA origami objects as rigid mech. mediators or force sensing elements requires detailed knowledge about their complex mech. properties. Using magnetic tweezers, the authors directly measure the bending and torsional rigidities of four- and six-helix bundles assembled by this technique. Compared to duplex DNA, the authors find the bending rigidities to be greatly increased while the torsional rigidities are only moderately augmented. The authors present a mech. model explicitly including the crossovers between the individual helixes in the origami structure that reproduces the exptl. obsd. behavior. The authors' results provide an important basis for the future application of 3D DNA origami in nanomechanics.
- 27Castro, C. E.; Su, H. J.; Marras, A. E.; Zhou, L.; Johnson, J. Mechanical Design of DNA Nanostructures. Nanoscale 2015, 7, 5913– 5921, DOI: 10.1039/C4NR07153KGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitlamsrc%253D&md5=eb12536fddc1a44a0e7fb7a44d38fcecMechanical design of DNA nanostructuresCastro, Carlos E.; Su, Hai-Jun; Marras, Alexander E.; Zhou, Lifeng; Johnson, JoshuaNanoscale (2015), 7 (14), 5913-5921CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)A review. Structural DNA nanotechnol. is a rapidly emerging field that has demonstrated great potential for applications such as single mol. sensing, drug delivery, and templating mol. components. As the applications of DNA nanotechnol. expand, a consideration of their mech. behavior is becoming essential to understand how these structures will respond to phys. interactions. This review considers three major avenues of recent progress in this area: (1) measuring and designing mech. properties of DNA nanostructures, (2) designing complex nanostructures based on imposed mech. stresses, and (3) designing and controlling structurally dynamic nanostructures. This work has laid the foundation for mech. active nanomachines that can generate, transmit, and respond to phys. cues in mol. systems.
- 28Buchkovich, N. J.; Henne, W. M.; Tang, S.; Emr, S. D. Essential N-Terminal Insertion Motif Anchors the Escrt-Iii Filament During Mvb Vesicle Formation. Dev. Cell 2013, 27, 201– 214, DOI: 10.1016/j.devcel.2013.09.009Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1CltrvM&md5=677ff08a5c363db1f11c8936e186e52dEssential N-Terminal Insertion Motif Anchors the ESCRT-III Filament during MVB Vesicle FormationBuchkovich, Nicholas J.; Henne, William Mike; Tang, Shaogeng; Emr, Scott D.Developmental Cell (2013), 27 (2), 201-214CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)The endosomal sorting complexes required for transport (ESCRTs) have emerged as key cellular machinery that drive topol. unique membrane deformation and scission. Understanding how the ESCRT-III polymer interacts with membrane, promoting and/or stabilizing membrane deformation, is an important step in elucidating this sculpting mechanism. Using a combination of genetic and biochem. approaches, both in vivo and in vitro, we identify two essential modules required for ESCRT-III-membrane assocn.: an electrostatic cluster and an N-terminal insertion motif. Mutating either module in yeast causes cargo sorting defects in the MVB pathway. We show that the essential N-terminal insertion motif provides a stable anchor for the ESCRT-III polymer. By replacing this N-terminal motif with well-characterized membrane insertion modules, we demonstrate that the N terminus of the Snf7 subunit has been tuned to maintain the topol. constraints assocd. with ESCRT-III-filament-mediated membrane invagination and vesicle formation. Our results provide insights into the spatially unique, ESCRT-III-mediated membrane remodeling.
- 29Kim, D. N.; Kilchherr, F.; Dietz, H.; Bathe, M. Quantitative Prediction of 3d Solution Shape and Flexibility of Nucleic Acid Nanostructures. Nucleic Acids Res. 2012, 40, 2862– 2868, DOI: 10.1093/nar/gkr1173Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xls1Snu7k%253D&md5=5570389892b346fe6198edd9361e21e1Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructuresKim, Do-Nyun; Kilchherr, Fabian; Dietz, Hendrik; Bathe, MarkNucleic Acids Research (2012), 40 (7), 2862-2868CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA nanotechnol. enables the programmed synthesis of intricate nanometer-scale structures for diverse applications in materials and biol. science. Precise control over the 3D soln. shape and mech. flexibility of target designs is important to achieve desired functionality. Because exptl. validation of designed nanostructures is time-consuming and cost-intensive, predictive phys. models of nanostructure shape and flexibility have the capacity to enhance dramatically the design process. Here, the authors significantly extend and exptl. validate a computational modeling framework for DNA origami previously presented as CanDo A primer to scaffolded DNA origami. 3D soln. shape and flexibility are predicted from basepair connectivity maps now accounting for nicks in the DNA double helix, entropic elasticity of single-stranded DNA, and distant crossovers required to model wireframe structures, in addn. to previous modeling that accounted only for the canonical twist, bend and stretch stiffness of double-helical DNA domains. Systematic exptl. validation of nanostructure flexibility mediated by internal crossover d. probed using a 32-helix DNA bundle demonstrates for the first time that our model not only predicts the 3D soln. shape of complex DNA nanostructures but also their mech. flexibility. Thus, our model represents an important advance in the quant. understanding of DNA-based nanostructure shape and flexibility, and we anticipate that this model will increase significantly the no. and variety of synthetic nanostructures designed using nucleic acids.
- 30Myhrvold, C.; Baym, M.; Hanikel, N.; Ong, L. L.; Gootenberg, J. S.; Yin, P. Barcode Extension for Analysis and Reconstruction of Structures. Nat. Commun. 2017, 8, 14698, DOI: 10.1038/ncomms14698Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXkslOjsLg%253D&md5=9bcd74283733debc855798ac30d4fceaBarcode extension for analysis and reconstruction of structuresMyhrvold, Cameron; Baym, Michael; Hanikel, Nikita; Ong, Luvena L.; Gootenberg, Jonathan S.; Yin, PengNature Communications (2017), 8 (), 14698CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Collections of DNA sequences can be rationally designed to self-assemble into predictable three-dimensional structures. The geometric and functional diversity of DNA nanostructures created to date has been enhanced by improvements in DNA synthesis and computational design. However, existing methods for structure characterization typically image the final product or laboriously det. the presence of individual, labeled strands using gel electrophoresis. Here we introduce a new method of structure characterization that uses barcode extension and next-generation DNA sequencing to quant. measure the incorporation of every strand into a DNA nanostructure. By quantifying the relative abundances of distinct DNA species in product and monomer bands, we can study the influence of geometry and sequence on assembly. We have tested our method using 2D and 3D DNA brick and DNA origami structures. Our method is general and should be extensible to a wide variety of DNA nanostructures.
- 31Benson, E.; Mohammed, A.; Rayneau-Kirkhope, D.; Gadin, A.; Orponen, P.; Hogberg, B. Effects of Design Choices on the Stiffness of Wireframe DNA Origami Structures. ACS Nano 2018, 12, 9291– 9299, DOI: 10.1021/acsnano.8b04148Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1Cns7nF&md5=edbea1de7199f7ac57283421cad4fedaEffects of Design Choices on the Stiffness of Wireframe DNA Origami StructuresBenson, Erik; Mohammed, Abdulmelik; Rayneau-Kirkhope, Daniel; Gaadin, Andreas; Orponen, Pekka; Hoegberg, BjoernACS Nano (2018), 12 (9), 9291-9299CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)DNA origami is a powerful method for the creation of 3D nanoscale objects, and in the past few years, interest in wireframe origami designs has increased due to their potential for biomedical applications. In DNA wireframe designs, the construction material is double-stranded DNA, which has a persistence length of around 50 nm. In this work, the authors study the effect of various design choices on the stiffness vs. final size of nanoscale wireframe rods, given the constraints on origami designs set by the DNA origami scaffold size. An initial theor. anal. predicts two competing mechanisms limiting rod stiffness, whose balancing results in an optimal edge length. For small edge lengths, the bending of the rod's overall frame geometry is the dominant factor, while the flexibility of individual DNA edges has a greater contribution at larger edge lengths. The authors evaluate the design choices through simulations and expts. and find that the stiffness of the structures increases with the no. of sides in the cross-section polygon and that there are indications of an optimal member edge length. The authors also ascertain the effect of nicked DNA edges on the stiffness of the wireframe rods and demonstrate that ligation of the staple breakpoint nicks reduces the obsd. flexibility. The simulations also indicate that the persistence length of wireframe DNA structures significantly decreases with increasing monovalent salt concn.
- 32Khmelinskaia, 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, DOI: 10.1088/0022-3727/49/19/194001Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFWrsb%252FK&md5=ea2abc7e490dd9b694d008f613daba67Effect of anchor positioning on binding and diffusion of elongated 3D DNA nanostructures on lipid membranesKhmelinskaia, Alena; Franquelim, Henri G.; Petrov, Eugene P.; Schwille, PetraJournal of Physics D: Applied Physics (2016), 49 (19), 194001/1-194001/11CODEN: JPAPBE; ISSN:0022-3727. (IOP Publishing Ltd.)DNA origami is a state-of-the-art technol. that enables the fabrication of nano-objects with defined shapes, to which functional moieties, such as lipophilic anchors, can be attached with a nanometer scale precision. Although binding of DNA origami to lipid membranes has been extensively demonstrated, the specific requirements necessary for membrane attachment are greatly overlooked. Here, we designed a set of amphipathic rectangular-shaped DNA origami structures with varying placement and no. of chol-TEG anchors used for membrane attachment. Single- and multiple-cholesteryl-modified origami nanostructures were produced and studied in terms of their membrane localization, d. and dynamics. We show that the positioning of at least two chol-TEG moieties near the corners is essential to ensure efficient membrane binding of large DNA nanostructures. Quant. fluorescence correlation spectroscopy data further confirm that increasing the no. of corner-positioned chol-TEG anchors lowers the dynamics of flat DNA origami structures on freestanding membranes. Taken together, our approach provides the first evidence of the importance of the location in addn. to the no. of hydrophobic moieties when rationally designing minimal DNA nanostructures with controlled membrane binding.
- 33Khmelinskaia, A.; Mucksch, J.; Petrov, E. P.; Franquelim, H. G.; Schwille, P. Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers. Langmuir 2018, 34, 14921– 14931, DOI: 10.1021/acs.langmuir.8b01850Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOqsrjE&md5=1df97032f81594bc11ff6ddb37d329f6Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA SpacersKhmelinskaia, Alena; Muecksch, Jonas; Petrov, Eugene P.; Franquelim, Henri G.; Schwille, PetraLangmuir (2018), 34 (49), 14921-14931CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)DNA origami nanotechnol. is increasingly used to mimic membrane-assocd. biophys. phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, the authors used a set of elongated DNA origami structures with varying placement and no. of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. The results show that the location and no. of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct phys. properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, the authors provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.
- 34Nguyen, N.; Shteyn, V.; Melia, T. J. Sensing Membrane Curvature in Macroautophagy. J. Mol. Biol. 2017, 429, 457– 472, DOI: 10.1016/j.jmb.2017.01.006Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVCgtLc%253D&md5=69b3124b6fbf60a64a84273150b3eda0Sensing membrane curvature in macroautophagyNguyen, Nathan; Shteyn, Vladimir; Melia, Thomas J.Journal of Molecular Biology (2017), 429 (4), 457-472CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)A review. In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo formation of a new organelle called the autophagosome, which either surrounds and sequesters random portions of the cytoplasm or selectively targets individual intracellular challenges. Thus, the autophagosome is a flexible membrane platform with dimensions that ultimately depend upon the target cargo. The intermediate membrane, termed the phagophore or isolation membrane, is a cup-like structure with a clear concave face and a highly curved rim. The phagophore is largely devoid of integral membrane proteins; thus, its shape and size are governed by peripherally assocd. membrane proteins and possibly by the lipid compn. of the membrane itself. Growth along the phagophore rim marks the progress of both organelle expansion and ultimately organelle closure around a particular cargo. These 2 properties, a reliance on peripheral membrane proteins and a structurally distinct membrane architecture, suggest that the ability to target or manipulate membrane curvature might be an essential activity of proteins functioning in this pathway. Here, the authors discuss the extent to which membranes are naturally curved at each of the cellular sites believed to engage in autophagosome formation, review basic mechanisms used to sense this curvature, and then summarize the existing literature concerning which autophagy proteins are capable of curvature recognition.
- 35Bhatia, V. K.; Hatzakis, N. S.; Stamou, D. A Unifying Mechanism Accounts for Sensing of Membrane Curvature by Bar Domains, Amphipathic Helices and Membrane-Anchored Proteins. Semin. Cell Dev. Biol. 2010, 21, 381– 390, DOI: 10.1016/j.semcdb.2009.12.004Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkvFKrt7o%253D&md5=d25e138be5fca2b7a8a235c938694f4cA unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteinsBhatia, Vikram Kjoeller; Hatzakis, Nikos S.; Stamou, DimitriosSeminars in Cell & Developmental Biology (2010), 21 (4), 381-390CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)A review. The discovery of proteins that recognize membrane curvature created a paradigm shift by suggesting that membrane shape may act as a cue for protein localization that is independent of lipid or protein compn. Here we review recent data on membrane curvature sensing by three structurally unrelated motifs: BAR domains, amphipathic helixes and membrane-anchored proteins. We discuss the conclusion that the curvature of the BAR dimer is not responsible for sensing and that the sensing properties of all three motifs can be rationalized by the physicochem. properties of the curved membrane itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biol.
- 36Douglas, S. M.; Marblestone, A. H.; Teerapittayanon, S.; Vazquez, A.; Church, G. M.; Shih, W. M. Rapid Prototyping of 3d DNA-Origami Shapes with Cadnano. Nucleic Acids Res. 2009, 37, 5001– 5006, DOI: 10.1093/nar/gkp436Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKntbzE&md5=aa99732c1666373a70e9b7b4de6e6d5dRapid prototyping of 3D DNA-origami shapes with caDNAnoDouglas, Shawn M.; Marblestone, Adam H.; Teerapittayanon, Surat; Vazquez, Alejandro; Church, George M.; Shih, William M.Nucleic Acids Research (2009), 37 (15), 5001-5006CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)DNA nanotechnol. exploits the programmable specificity afforded by base-pairing to produce self-assembling macromol. objects of custom shape. For building megadalton-scale DNA nanostructures, a long scaffold' strand can be employed to template the assembly of hundreds of oligonucleotide staple' strands into a planar antiparallel array of cross-linked helixes. The authors recently adapted this scaffolded DNA origami' method to producing 3-dimensional shapes formed as pleated layers of double helixes constrained to a honeycomb lattice. However, completing the required design steps can be cumbersome and time-consuming. Here the authors present caDNAno, an open-source software package with a graphical user interface that aids in the design of DNA sequences for folding 3-dimensional honeycomb-pleated shapes rectangular-block motifs were designed, assembled, and analyzed to identify a well-behaved motif that could serve as a building block for future studies. The use of caDNAno significantly reduces the effort required to design 3-dimensional DNA-origami structures. The software is available at http://cadnano.org/, along with example designs and video tutorials demonstrating their construction. The source code is released under the MIT license.
- 37Bellot, G.; McClintock, M. A.; Chou, J. J.; Shih, W. M. DNA Nanotubes for Nmr Structure Determination of Membrane Proteins. Nat. Protoc. 2013, 8, 755– 770, DOI: 10.1038/nprot.2013.037Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXmtlShsbo%253D&md5=21847aedab3e0bcecdca6028ac8c92deDNA nanotubes for NMR structure determination of membrane proteinsBellot, Gaetan; McClintock, Mark A.; Chou, James J.; Shih, William M.Nature Protocols (2013), 8 (4), 755-770, 16 pp.CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Finding a way to det. the structures of integral membrane proteins using soln. NMR (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dil. liq. crystals for application as weak-alignment media in soln. NMR structure detn. of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable pos. charge as well as modulation of mol. alignment, toward collection of linearly independent restraints, can be introduced by reducing the neg. charge of DNA nanotubes using counter ions and small DNA-binding mols. This detergent-resistant liq.-crystal medium offers a no. of properties conducive for membrane protein alignment, including high-yield prodn., thermal stability, buffer compatibility and structural programmability. Prodn. of sufficient nanotubes for four or five NMR expts. can be completed in 1 wk by a single individual.
- 38Lin, C.; Perrault, S. D.; Kwak, M.; Graf, F.; Shih, W. M. Purification of DNA-Origami Nanostructures by Rate-Zonal Centrifugation. Nucleic Acids Res. 2013, 41, e40, DOI: 10.1093/nar/gks1070Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtFyjurg%253D&md5=55de3fad844f9e5e0802d5abca92f5afPurification of DNA-origami nanostructures by rate-zonal centrifugationLin, Chenxiang; Perrault, Steven D.; Kwak, Minseok; Graf, Franziska; Shih, William M.Nucleic Acids Research (2013), 41 (2), e40CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Most previously reported methods for purifying DNA-origami nanostructures have relied on agarose-gel electrophoresis (AGE) for sepn. Although AGE is routinely used to yield 0.1-1 μg purified DNA nanostructures, obtaining >100 μg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extn., desalting, and concn. steps. Here, the authors present a readily scalable purifn. approach utilizing rate-zonal centrifugation, which provides comparable sepn. resoln. as AGE. The DNA nanostructures remain in aq. soln. throughout the purifn. process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA-intercalating dyes. Seven distinct 3-dimensional DNA-origami constructs were purified at the scale of 0.1-100 μg (final yield) per centrifuge tube, showing the versatility of this method. Given the com. available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for prepn. of larger amts. (e.g., milligram quantities) of DNA nanostructures.
- 39Stahl, E.; Martin, T. G.; Praetorius, F.; Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angew. Chem., Int. Ed. 2014, 53, 12735– 12740, DOI: 10.1002/anie.201405991Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvV2mtrrM&md5=5d749b225ce5405de5c89eeb1a5b1897Facile and Scalable Preparation of Pure and Dense DNA Origami SolutionsStahl, Evi; Martin, Thomas G.; Praetorius, Florian; Dietz, HendrikAngewandte Chemie, International Edition (2014), 53 (47), 12735-12740CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)DNA has become a prime material for assembling complex three-dimensional objects that promise utility in various areas of application. However, achieving user-defined goals with DNA objects has been hampered by the difficulty to prep. them at arbitrary concns. and in user-defined soln. conditions. Here, we describe a method that solves this problem. The method is based on poly(ethylene glycol)-induced depletion of species with high mol. wt. We demonstrate that our method is applicable to a wide spectrum of DNA shapes and that it achieves excellent recovery yields of target objects up to 97 %, while providing efficient sepn. from non-integrated DNA strands. DNA objects may be prepd. at concns. up to the limit of soly., including the possibility for bringing DNA objects into a solid phase. Due to the fidelity and simplicity of our method we anticipate that it will help to catalyze the development of new types of applications that use self-assembled DNA objects.
- 40Lamour, G.; Kirkegaard, J. B.; Li, H.; Knowles, T. P.; Gsponer, J. Easyworm: An Open-Source Software Tool to Determine the Mechanical Properties of Worm-Like Chains. Source Code Biol. Med. 2014, 9, 16, DOI: 10.1186/1751-0473-9-16Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cbptVOhtQ%253D%253D&md5=6462166224a41bc00fd68752c3cc6a63Easyworm: an open-source software tool to determine the mechanical properties of worm-like chainsLamour Guillaume; Kirkegaard Julius B; Knowles Tuomas Pj; Li Hongbin; Gsponer JorgSource code for biology and medicine (2014), 9 (), 16 ISSN:1751-0473.BACKGROUND: A growing spectrum of applications for natural and synthetic polymers, whether in industry or for biomedical research, demands for fast and universally applicable tools to determine the mechanical properties of very diverse polymers. To date, determining these properties is the privilege of a limited circle of biophysicists and engineers with appropriate technical skills. FINDINGS: Easyworm is a user-friendly software suite coded in MATLAB that simplifies the image analysis of individual polymeric chains and the extraction of the mechanical properties of these chains. Easyworm contains a comprehensive set of tools that, amongst others, allow the persistence length of single chains and the Young's modulus of elasticity to be calculated in multiple ways from images of polymers obtained by a variety of techniques (e.g. atomic force microscopy, electron, contrast-phase, or epifluorescence microscopy). CONCLUSIONS: Easyworm thus provides a simple and efficient tool for specialists and non-specialists alike to solve a common problem in (bio)polymer science. Stand-alone executables and shell scripts are provided along with source code for further development.
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ARTICLE SECTIONSThe Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05401.
Designs of DNA-origami curls, DNA sequences, materials and methods, and additional data and models (PDF)
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