Protein-Assisted Large-Scale Assembly and Differential Patterning of DNA Origami Lattices.

Nanofabrication has experienced a big boost with the invention of DNA origami, enabling the production and assembly of complex nanoscale structures that may be able to unlock fully new functionalities in biology and beyond. The remarkable precision with which these structures can be designed and produced is, however, not yet matched by their assembly dynamics, which can be extremely slow, particularly when attached to biological templates, such as membranes. Here, the rapid and controlled formation of DNA origami lattices on the scale of hundreds of micrometers in as little as 30 minutes is demonstrated, utilizing active patterning by the E.coli Min protein system, thereby yielding a remarkable improvement over conventional passive diffusion-based assembly methods. Various patterns, including spots, inverse spots, mazes, and meshes can be produced at different scales, tailored through the shape and density of the assembled structures. The differential positioning accomplished by Min-induced diffusiophoresis even allows the introduction of "pseudo-colors", i.e., complex core-shell patterns, by simultaneously patterning different DNA origami species. Beyond the targeted functionalization of biological surfaces, this approach may also be promising for applications in plasmonics, catalysis, and molecular sensing.

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release.

Doxorubicin (DOX) is a common drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of DOX-loaded DNA-carriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures (DONs). In our experimental conditions, all DONs show similar DOX binding capacities (one DOX molecule per two to three base pairs), and the binding equilibrium is reached within seconds, remarkably faster than previously acknowledged. To characterize drug release profiles, DON degradation and DOX release from the complexes upon DNase I digestion was studied. For the employed DONs, the relative doses (DOX molecules released per unit time) may vary by two orders of magnitude depending on the DON superstructure. In addition, we identify DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration. These features have been largely ignored in experimenting with DNA nanostructures, but are probably the major sources of the incoherence of the experimental results so far. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA-carriers.

Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells.

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100× more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses.

The Role of Ligands in the Chemical Synthesis and Applications of Inorganic Nanoparticles.

The design of nanoparticles is critical for their efficient use in many applications ranging from biomedicine to sensing and energy. While shape and size are responsible for the properties of the inorganic nanoparticle core, the choice of ligands is of utmost importance for the colloidal stability and function of the nanoparticles. Moreover, the selection of ligands employed in nanoparticle synthesis can determine their final size and shape. Ligands added after nanoparticle synthesis infer both new properties as well as provide enhanced colloidal stability. In this article, we provide a comprehensive review on the role of the ligands with respect to the nanoparticle morphology, stability, and function. We analyze the interaction of nanoparticle surface and ligands with different chemical groups, the types of bonding, the final dispersibility of ligand-coated nanoparticles in complex media, their reactivity, and their performance in biomedicine, photodetectors, photovoltaic devices, light-emitting devices, sensors, memory devices, thermoelectric applications, and catalysis.

Cryopreservation of DNA Origami Nanostructures.

Although DNA origami nanostructures have found their way into numerous fields of fundamental and applied research, they often suffer from rather limited stability when subjected to environments that differ from the employed assembly conditions, that is, suspended in Mg2+ -containing buffer at moderate temperatures. Here, means for efficient cryopreservation of 2D and 3D DNA origami nanostructures and, in particular, the effect of repeated freezing and thawing cycles are investigated. It is found that, while the 2D DNA origami nanostructures maintain their structural integrity over at least 32 freeze-thaw cycles, ice crystal formation makes the DNA origami gradually more sensitive toward harsh sample treatment conditions. Whereas no freeze damage could be detected in 3D DNA origami nanostructures subjected to 32 freeze-thaw cycles, 1000 freeze-thaw cycles result in significant fragmentation. The cryoprotectants glycerol and trehalose are found to efficiently protect the DNA origami nanostructures against freeze damage at concentrations between 0.2 × 10-3 and 200 × 10-3 m and without any negative effects on DNA origami shape. This work thus provides a basis for the long-term storage of DNA origami nanostructures, which is an important prerequisite for various technological and medical applications.

DNA-Origami-Templated Silica Growth by Sol-Gel Chemistry.

Improving the stability of DNA origami structures with respect to thermal, chemical, and mechanical demands will be essential to fully explore the real-life applicability of DNA nanotechnology. Here we present a strategy to increase the mechanical resilience of individual DNA origami objects and 3D DNA origami crystals in solution as well as in the dry state. By encapsulating DNA origami in a protective silica shell using sol-gel chemistry, all the objects maintain their structural integrity. This allowed for a detailed structural analysis of the crystals in a dry state, thereby revealing their true 3D shape without lattice deformation and drying-induced collapse. Analysis by energy-dispersive X-ray spectroscopy showed a uniform silica coating whose thickness could be controlled through the precursor concentrations and reaction time. This strategy thus facilitates shape-controlled bottom-up synthesis of designable biomimetic silica structures through transcription from DNA origami.

Sensing of Vimentin mRNA in 2D and 3D Models of Wounded Skin Using DNA-Coated Gold Nanoparticles.

Wound healing is a highly complex biological process, which is accompanied by changes in cell phenotype, variations in protein expression, and the production of active biomolecules. Currently, the detection of proteins in cells is done by immunostaining where the proteins in fixed cells are detected by labeled antibodies. However, immunostaining cannot provide information about dynamic processes in living cells, within the whole tissue. Here, an easy method is presented to detect the transition of epithelial to mesenchymal cells during wound healing. The method employs DNA-coated gold nanoparticle fluorescent nanoprobes to sense the production of Vimentin mRNA expressed in mesenchymal cells. Fluorescence microscopy is used to achieve temporal detection of Vimentin mRNA in wounds. 3D light-sheet microscopy is utilized to observe the dynamic expression of Vimentin mRNA spatially around the wounded site in skin tissue. The use of DNA-gold nanoprobes to detect mRNA expression during wound healing opens up new possibilities for the study of real-time mechanisms in complex biological processes.

Potentiating angiogenesis arrest in vivo via laser irradiation of peptide functionalised gold nanoparticles.

BACKGROUND: Anti-angiogenic therapy has great potential for cancer therapy with several FDA approved formulations but there are considerable side effects upon the normal blood vessels that decrease the potential application of such therapeutics. Chicken chorioallantoic membrane (CAM) has been used as a model to study angiogenesis in vivo. Using a CAM model, it had been previously shown that spherical gold nanoparticles functionalised with an anti-angiogenic peptide can humper neo-angiogenesis. RESULTS: Our results show that gold nanoparticles conjugated with an anti-angiogenic peptide can be combined with visible laser irradiation to enhance angiogenesis arrest in vivo. We show that a green laser coupled to gold nanoparticles can achieve high localized temperatures able to precisely cauterize blood vessels. This combined therapy acts via VEGFR pathway inhibition, leading to a fourfold reduction in FLT-1 expression. CONCLUSIONS: The proposed phototherapy extends the use of visible lasers in clinics, combining it with chemotherapy to potentiate cancer treatment. This approach allows the reduction of dose of anti-angiogenic peptide, thus reducing possible side effects, while destroying blood vessels supply critical for tumour progression.

Interactions of skin with gold nanoparticles of different surface charge, shape, and functionality.

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP-OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R7 are found in the skin in larger quantities than polyethylene glycol-functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.

Lyophilization Reduces Aggregation of Three-Dimensional DNA Origami at High Concentrations.

Although for many purposes, low concentrations of DNA origami are sufficient, certain applications such as cryo electron microscopy, measurements involving small-angle X-ray scattering, or in vivo applications require high DNA origami concentrations of >200 nM. This is achievable by ultrafiltration or polyethylene glycol precipitation but often at the expense of increasing structural aggregation due to prolonged centrifugation and final redispersion in low buffer volumes. Here, we show that lyophilization and subsequent redispersion in low buffer volumes can achieve high concentrations of DNA origami while drastically reducing aggregation due to initially very low DNA origami concentrations in low salt buffers. We demonstrate this for four structurally different types of three-dimensional DNA origami. All of these structures exhibit different aggregation behaviors at high concentrations (tip-to-tip stacking, side-to-side binding, or structural interlocking), which can be drastically reduced by dispersion in larger volumes of a low salt buffer and subsequent lyophilization. Finally, we show that this procedure can also be applied to silicified DNA origami to achieve high concentrations with low aggregation. We thus find that lyophilization is not only a tool for long-term storage of biomolecules but also an excellent way for up-concentrating while maintaining well-dispersed solutions of DNA origami.

Full Site-Specific Addressability in DNA Origami-Templated Silica Nanostructures.

DNA nanotechnology allows for the fabrication of nanometer-sized objects with high precision and selective addressability as a result of the programmable hybridization of complementary DNA strands. Such structures can template the formation of other materials, including metals and complex silica nanostructures, where the silica shell simultaneously acts to protect the DNA from external detrimental factors. However, the formation of silica nanostructures with site-specific addressability has thus far not been explored. Here, it is shown that silica nanostructures templated by DNA origami remain addressable for post silicification modification with guest molecules even if the silica shell measures several nm in thickness. The conjugation of fluorescently labeled oligonucleotides is used to different silicified DNA origami structures carrying a complementary ssDNA handle as well as DNA-PAINT super-resolution imaging to show that ssDNA handles remain unsilicified and thus ensure retained addressability. It is also demonstrated that not only handles, but also ssDNA scaffold segments within a DNA origami nanostructure remain accessible, allowing for the formation of dynamic silica nanostructures. Finally, the power of this approach is demonstrated by forming 3D DNA origami crystals from silicified monomers. These results thus present a fully site-specifically addressable silica nanostructure with complete control over size and shape.

In situ small-angle X-ray scattering reveals strong condensation of DNA origami during silicification.

Silicification of DNA origami structures increases their stability and provides chemical protection. Yet, it is unclear whether the whole DNA framework is embedded or if silica just forms an outer shell and how silicification affects the origami's internal structure. Employing in situ small-angle X-ray scattering (SAXS), we show that addition of silica precursors induces substantial condensation of the DNA origami at early reaction times by almost 10 %. Subsequently, the overall size of the silicified DNA origami increases again due to increasing silica deposition. We further identify the SAXS Porod invariant as a reliable, model-free parameter for the evaluation of the amount of silica formation at a given time. Contrast matching of the DNA double helix Lorentzian peak reveals silica growth also inside the origami. The less polar silica forming within the origami structure, replacing more than 40 % of the internal hydration water, causes a hydrophobic effect: condensation. DNA origami objects with flat surfaces show a strong tendency towards aggregation during silicification, presumably driven by the same entropic forces causing condensation. Maximally condensed origami displayed thermal stability up to 60 °C. Our studies provide insights into the silicification reaction allowing for the formulation of optimized reaction protocols.

Engineering Inorganic Materials with DNA Nanostructures.

Nucleic acid nanotechnology lays a foundation for the user-friendly design and synthesis of DNA frameworks of any desirable shape with extreme accuracy and addressability. Undoubtedly, such features make these structures ideal modules for positioning and organizing molecules and molecular components into complex assemblies. One of the emerging concepts in the field is to create inorganic and hybrid materials through programmable DNA templates. Here, we discuss the challenges and perspectives of such DNA nanostructure-driven materials science engineering and provide insights into the subject by introducing various DNA-based fabrication techniques including metallization, mineralization, lithography, casting, and hierarchical self-assembly of metal nanoparticles.

Chiral Assembly of Gold-Silver Core-Shell Plasmonic Nanorods on DNA Origami with Strong Optical Activity.

The spatial organization of metal nanoparticles has become an important tool for manipulating light in nanophotonic applications. Silver nanoparticles, particularly silver nanorods, have excellent plasmonic properties but are prone to oxidation and are therefore inherently unstable in aqueous solutions and salt-containing buffers. Consequently, gold nanoparticles have often been favored, despite their inferior optical performance. Bimetallic, i.e., gold-silver core-shell nanoparticles, can resolve this issue. We present a method for synthesizing highly stable gold-silver core-shell NRs that are instantaneously functionalized with DNA, enabling chiral self-assembly on DNA origami. The silver shell gives rise to an enhancement of plasmonic properties, reflected here in strongly increased circular dichroism, as compared to pristine gold nanorods. Gold-silver nanorods are ideal candidates for plasmonic sensing with increased sensitivity as needed in pathogen RNA or antibody testing for nonlinear optics and light-funneling applications in surface enhanced Raman spectroscopy. Furthermore, the control of interparticle orientation enables the study of plasmonic phenomena, in particular, synergistic effects arising from plasmonic coupling of such bimetallic systems.

3D DNA Origami Crystals.

3D crystals assembled entirely from DNA provide a route to design materials on a molecular level and to arrange guest particles in predefined lattices. This requires design schemes that provide high rigidity and sufficiently large open guest space. A DNA-origami-based "tensegrity triangle" structure that assembles into a 3D rhombohedral crystalline lattice with an open structure in which 90% of the volume is empty space is presented here. Site-specific placement of gold nanoparticles within the lattice demonstrates that these crystals are spacious enough to efficiently host 20 nm particles in a cavity size of 1.83 × 105 nm3 , which would also suffice to accommodate ribosome-sized macromolecules. The accurate assembly of the DNA origami lattice itself, as well as the precise incorporation of gold particles, is validated by electron microscopy and small-angle X-ray scattering experiments. The results show that it is possible to create DNA building blocks that assemble into lattices with customized geometry. Site-specific hosting of nano objects in the optically transparent DNA lattice sets the stage for metamaterial and structural biology applications.

Cells on hierarchically-structured platforms hosting functionalized nanoparticles.

In this work, we report on a novel approach to develop hierarchically-structured cell culture platforms incorporating functionalized gold nanoparticles (AuNPs). In particular, the hierarchical substrates comprise primary pseudo-periodic arrays of silicon microcones combined with a secondary nanoscale pattern of homogeneously deposited AuNPs terminated with bio-functional moieties. AuNPs with various functionalities (i.e. oligopeptides, small molecules and oligomers) were successfully attached onto the microstructures. Experiments with PC12 cells on hierarchical substrates incorporating AuNPs carrying the RGD peptide showed an impressive growth and NGF-induced differentiation of the PC12 cells, compared to that on the NP-free, bare, micropatterned substrates. The exploitation of the developed methodology for the binding of AuNPs as carriers of specific bio-functional moieties onto micropatterned culture substrates for cell biology studies is envisaged.

Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo.

In this work, peptides designed to selectively interact with cellular receptors involved in the regulation of angiogenesis were anchored to oligo-ethylene glycol-capped gold nanoparticles (AuNPs) and used to evaluate the modulation of vascular development using an ex ovo chick chorioallantoic membrane assay. These nanoparticles alter the balance between naturally secreted pro- and antiangiogenic factors, under various biological conditions, without causing toxicity. Exposure of chorioallantoic membranes to AuNP-peptide activators of angiogenesis accelerated the formation of new arterioles when compared to scrambled peptide-coated nanoparticles. On the other hand, antiangiogenic AuNP-peptide conjugates were able to selectively inhibit angiogenesis in vivo. We demonstrated that AuNP vectorization is crucial for enhancing the effect of active peptides. Our data showed for the first time the effective control of activation or inhibition of blood vessel formation in chick embryo via AuNP-based formulations suitable for the selective modulation of angiogenesis, which is of paramount importance in applications where promotion of vascular growth is desirable (eg, wound healing) or ought to be contravened, as in cancer development.

Selective killing of cells triggered by their mRNA signature in the presence of smart nanoparticles.

The design of nanoparticles that can selectively perform multiple roles is of utmost importance for the development of the next generation of nanoparticulate drug delivery systems. So far most research studies are focused on the customization of nanoparticulate carriers to maximize their drug loading, enhance their optical signature for tracking in cells or provide photo-responsive effects for therapeutic purposes. However, a vital requirement of the new generation of drug carriers must be the ability to deliver their payload selectively only to cells of interest rather than the majority of various cells in the vicinity. Here we show for the first time a new design of nanoparticulate drug carriers that can specifically distinguish different cell types based on their mRNA signature. These nanoparticles sense and efficiently kill model tumour cells by the delivery of an anti-cancer drug but retain their payload in cells lacking the specific mRNA target.

Polymer-Enhanced Stability of Inorganic Perovskite Nanocrystals and Their Application in Color Conversion LEDs.

Cesium lead halide (CsPbX3, X = Cl, Br, I) nanocrystals (NCs) offer exceptional optical properties for several potential applications but their implementation is hindered by a low chemical and structural stability and limited processability. In the present work, we developed a new method to efficiently coat CsPbX3 NCs, which resulted in their increased chemical and optical stability as well as processability. The method is based on the incorporation of poly(maleic anhydride-alt-1-octadecene) (PMA) into the synthesis of the perovskite NCs. The presence of PMA in the ligand shell stabilizes the NCs by tightening the ligand binding, limiting in this way the NC surface interaction with the surrounding media. We further show that these NCs can be embedded in self-standing silicone/glass plates as down-conversion filters for the fabrication of monochromatic green and white light emitting diodes (LEDs) with narrow bandwidths and appealing color characteristics.

Copper-free click chemistry as an emerging tool for the programmed ligation of DNA-functionalised gold nanoparticles.

We demonstrate a new method to program the ligation of single stranded DNA-modified gold nanoparticles using copper-free click chemistry. Gold nanoparticles functionalized with a discrete number of 3'-azide or 5'-alkyne modified oligonucleotides, can be brought together via a splint strand and covalently 'clicked', in a simple one-pot reaction. This new approach to the assembly of gold nanoparticles is inherently advantageous in comparison to the traditional enzymatic ligation. The chemical ligation is specific and takes place at room temperature by simply mixing the particles without the need for special enzymatic conditions. The yield of 'clicked' nanoparticles can be as high as 92%. The ease of the copper-free, 'click-ligation' method allows for its universal applicability and opens up new avenues in programmed nanoparticle organization.