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19 pages, 44655 KiB

Open AccessArticle

Programmed Self-Assembly of DNA Nanosheets with Discrete Single-Molecule Thickness and Interfacial Mechanics: Design, Simulation, and Characterization

by Keitel Cervantes-Salguero, Yair Augusto Gutiérrez Fosado, William Megone, Julien E. Gautrot and Matteo Palma

Molecules 2023, 28(9), 3686; https://doi.org/10.3390/molecules28093686 - 24 Apr 2023

Cited by 2 | Viewed by 2132

Abstract

DNA is programmed to hierarchically self-assemble into superstructures spanning from nanometer to micrometer scales. Here, we demonstrate DNA nanosheets assembled out of a rationally designed flexible DNA unit (F-unit), whose shape resembles a Feynman diagram. F-units were designed to self-assemble in two dimensions [...] Read more.

DNA is programmed to hierarchically self-assemble into superstructures spanning from nanometer to micrometer scales. Here, we demonstrate DNA nanosheets assembled out of a rationally designed flexible DNA unit (F-unit), whose shape resembles a Feynman diagram. F-units were designed to self-assemble in two dimensions and to display a high DNA density of hydrophobic moieties. oxDNA simulations confirmed the planarity of the F-unit. DNA nanosheets with a thickness of a single DNA duplex layer and with large coverage (at least 30 μm × 30 μm) were assembled from the liquid phase at the solid/liquid interface, as unambiguously evidenced by atomic force microscopy imaging. Interestingly, single-layer nanodiscs formed in solution at low DNA concentrations. DNA nanosheet superstructures were further assembled at liquid/liquid interfaces, as demonstrated by the fluorescence of a double-stranded DNA intercalator. Moreover, the interfacial mechanical properties of the nanosheet superstructures were measured as a response to temperature changes, demonstrating the control of interfacial shear mechanics based on DNA nanostructure engineering. The rational design of the F-unit, along with the presented results, provide an avenue toward the controlled assembly of reconfigurable/responsive nanosheets and membranes at liquid/liquid interfaces, to be potentially used in the characterization of biomechanical processes and materials transport. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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14 pages, 23593 KiB

Open AccessArticle

Active Self-Assembly of Ladder-Shaped DNA Carrier for Drug Delivery

by Yuan Liu, Jiaxin Wang, Lijun Sun, Bin Wang, Qiang Zhang, Xiaokang Zhang and Ben Cao

Molecules 2023, 28(2), 797; https://doi.org/10.3390/molecules28020797 - 13 Jan 2023

Viewed by 2156

Abstract

With the advent of nanotechnology, DNA molecules have been transformed from solely genetic information carriers to multifunctional materials, showing a tremendous potential for drug delivery and disease diagnosis. In drug delivery systems, DNA is used as a building material to construct drug carriers [...] Read more.

With the advent of nanotechnology, DNA molecules have been transformed from solely genetic information carriers to multifunctional materials, showing a tremendous potential for drug delivery and disease diagnosis. In drug delivery systems, DNA is used as a building material to construct drug carriers through a variety of DNA self-assembly methods, which can integrate multiple functions to complete in vivo and in situ tasks. In this study, ladder-shaped drug carriers are developed for drug delivery on the basis of a DNA nanoladder. We first demonstrate the overall structure of the nanoladder, in which a nick is added into each rung of the nanoladder to endow the nanoladder with the ability to incorporate a drug loading site. The structure is designed to counteract the decrement of stability caused by the nick and investigated in different conditions to gain insight into the properties of the nicked DNA nanoladders. As a proof of concept, we fix the biotin in every other nick as a loading site and assemble the protein (streptavidin) on the loading site to demonstrate the feasibility of the drug-carrying function. The protein can be fixed stably and can be extended to different biological and chemical drugs by altering the drug loading site. We believe this design approach will be a novel addition to the toolbox of DNA nanotechnology, and it will be useful for versatile applications such as in bioimaging, biosensing, and targeted therapy. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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14 pages, 3314 KiB

Open AccessArticle

Catalytic RNA Oligomers Formed by Co-Oligomerization of a Pair of Bimolecular RNase P Ribozymes

by Mst. Ayesha Siddika, Takahiro Yamada, Risako Aoyama, Kumi Hidaka, Hiroshi Sugiyama, Masayuki Endo, Shigeyoshi Matsumura and Yoshiya Ikawa

Molecules 2022, 27(23), 8298; https://doi.org/10.3390/molecules27238298 - 28 Nov 2022

Cited by 1 | Viewed by 1451

Abstract

Naturally occurring ribozymes with a modular architecture are promising platforms for construction of RNA nanostructures because modular redesign enables their oligomerization. The resulting RNA nanostructures can exhibit the catalytic function of the parent ribozyme in an assembly dependent manner. In this study, we [...] Read more.

Naturally occurring ribozymes with a modular architecture are promising platforms for construction of RNA nanostructures because modular redesign enables their oligomerization. The resulting RNA nanostructures can exhibit the catalytic function of the parent ribozyme in an assembly dependent manner. In this study, we designed and constructed open-form oligomers of a bimolecular form of an RNase P ribozyme. The ribozyme oligomers were analyzed biochemically and by atomic force microscopy (AFM). Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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15 pages, 2429 KiB

Open AccessArticle

Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence

by Abimbola F. Adedeji Olulana, Dianne Choi, Vincent Inverso, Shiv K. Redhu, Marco Vidonis, Luca Crevatin, Allen W. Nicholson and Matteo Castronovo

Molecules 2022, 27(16), 5262; https://doi.org/10.3390/molecules27165262 - 17 Aug 2022

Cited by 1 | Viewed by 1763

Abstract

Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour [...] Read more.

Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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15 pages, 4112 KiB

Open AccessArticle

Selective Integrin α₅β₁ Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles

by Eva E. Kurisinkal, Vincenzo Caroprese, Marianna M. Koga, Diana Morzy and Maartje M. C. Bastings

Molecules 2022, 27(15), 4968; https://doi.org/10.3390/molecules27154968 - 4 Aug 2022

Cited by 3 | Viewed by 2613

Abstract

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and [...] Read more.

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α₅β₁ clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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12 pages, 10490 KiB

Open AccessArticle

A Surfactant Enables Efficient Membrane Spanning by Non-Aggregating DNA-Based Ion Channels

by Diana Morzy, Michael Schaich and Ulrich F. Keyser

Molecules 2022, 27(2), 578; https://doi.org/10.3390/molecules27020578 - 17 Jan 2022

Cited by 7 | Viewed by 3065

Abstract

DNA nanotechnology makes use of hydrophobically modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by [...] Read more.

DNA nanotechnology makes use of hydrophobically modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by disrupting hydrophobicity-mediated clusters. Here, we employed confocal microscopy and single-molecule transmembrane current measurements to assess the effects of the non-ionic surfactant octylpolyoxyethylene (oPOE) on the clustering behavior and membrane activity of cholesterol-modified DNA nanostructures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydrophobically decorated constructs, and we highlight that premixing DNA structures with the surfactant does not disrupt the cholesterol-mediated aggregates. However, we observed the surfactant’s strong insertion-facilitating effect, particularly when introduced to the sample separately from DNA. Critically, we report a highly efficient membrane-spanning DNA construct from combining a non-aggregating design with the addition of the oPOE surfactant. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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16 pages, 7644 KiB

Open AccessArticle

Magnesium-Free Immobilization of DNA Origami Nanostructures at Mica Surfaces for Atomic Force Microscopy

by Yang Xin, Amir Ardalan Zargariantabrizi, Guido Grundmeier and Adrian Keller

Molecules 2021, 26(16), 4798; https://doi.org/10.3390/molecules26164798 - 7 Aug 2021

Cited by 8 | Viewed by 3865

Abstract

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg²⁺ ions to the sample [...] Read more.

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg²⁺ ions to the sample solution, which enable the adsorption of the negatively charged DONs at the like-charged mica surface. These non-physiological Mg²⁺ concentrations, however, present a serious limitation in such experiments as they may interfere with the reactions and processes under investigation. Therefore, we here evaluate three approaches to efficiently immobilize DONs at mica surfaces under essentially Mg²⁺-free conditions. These approaches rely on the pre-adsorption of different multivalent cations, i.e., Ni²⁺, poly-l-lysine (PLL), and spermidine (Spdn). DON adsorption is studied in phosphate-buffered saline (PBS) and pure water. In general, Ni²⁺ shows the worst performance with heavily deformed DONs. For 2D DON triangles, adsorption at PLL- and in particular Spdn-modified mica may outperform even Mg²⁺-mediated adsorption in terms of surface coverage, depending on the employed solution. For 3D six-helix bundles, less pronounced differences between the individual strategies are observed. Our results provide some general guidance for the immobilization of DONs at mica surfaces under Mg²⁺-free conditions and may aid future in situ AFM studies. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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Review

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10 pages, 4600 KiB

Open AccessReview

Surface Assembly of DNA Origami on a Lipid Bilayer Observed Using High-Speed Atomic Force Microscopy

by Masayuki Endo

Molecules 2022, 27(13), 4224; https://doi.org/10.3390/molecules27134224 - 30 Jun 2022

Cited by 6 | Viewed by 2413

Abstract

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on [...] Read more.

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice–tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture. Full article

(This article belongs to the Special Issue DNA Nanostructures at Surfaces)

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