Block Copolymer Micellization of DNA Origami Promotes Solubility in Organic Solvents.

The DNA origami technique allows the precise synthesis of complex, biocompatible nanomaterials containing small molecules, biomolecules, and inorganic nanoparticles. The negatively charged phosphates in the backbone make DNA highly water-soluble and require salts to shield its electrostatic repulsion. DNA origamis are therefore not soluble in most organic solvents. While this is not problematic for applications in biochemistry, biophysics, or nanomedicine, other potential applications, processes, and substrates are incompatible with saline solutions, which include the synthesis of many nanomaterials, and reactions in templated synthesis, the operation of nanoelectronic devices, or semiconductor fabrication. To overcome this limitation, we coated DNA origami with amphiphilic poly(ethylene glycol) polylysine block copolymers and transferred them into various organic solvents including chloroform, dichloromethane, acetone, or 1-propanol. Our approach maintains the shape of the nanostructures and protects functional elements bound to the structure, such as fluorophores, gold nanoparticles, or proteins. The DNA origami polyplex micellization (DOPM) strategy hence enables solubilization or a phase transfer of complex structures into various organic solvents, which significantly expands the use of DNA origami for a range of potential applications and technical processes.

Rolling circle amplification shows a sinusoidal template length-dependent amplification bias.

Biophysical properties of DNA such as its longitudinal and torsional persistence length govern many processes and phenomena in biology, DNA nanotechnology and biotechnology. It has, for example, long been known that the circularization efficiency of short DNA fragments shows a periodic pattern where fragments with integer helical turns circularize much more efficiently than those with odd helical half turns due to stronger stacking of duplex ends. Small DNA circles can serve as templates for rolling circle amplification (RCA), which is a common and extremely robust amplification mechanism for nucleic acids. We discovered a strong template length-dependent amplification efficiency bias of RCA with the same periodicity as B-DNA. However, stacking cannot explain the mechanism behind this bias as the presence of the polymerase in the bifurcation fork inhibits base stacking of ends. Instead, coarse-grained molecular dynamics simulations imply that different amplification efficiencies come from a varying fraying probability of the last two downstream base pairs. We conclude that an increased strain-promoted fraying probability can increase the polymerization rate compared to a relaxed template.

Highly Localized SERS Measurements Using Single Silicon Nanowires Decorated with DNA Origami-Based SERS Probe.

Surface enhanced Raman spectroscopy (SERS) measurements are conventionally performed using assemblies of metal nanostructures on a macro- to micro-sized substrate or by dispersing colloidal metal nanoparticles directly onto the sample of interest. Despite intense use, these methods allow neither the removal of the nanoparticles after a measurement nor a defined confinement of the SERS measurement position. So far, tip enhanced Raman spectroscopy is still the key technique in this regard but not adequate for various samples mainly due to diminished signal enhancement compared to other techniques, poor device fabrication reproducibility, and cumbersome experimental setup requirements. Here, we demonstrate that a rational combination of only four gold nanoparticles (AuNPs) on a DNA origami template, and single silicon nanowires (SiNWs) yield functional optical amplifier nanoprobes for SERS. These nanoscale SERS devices offer a spatial resolution below the diffraction limit of light and still a high electric field intensity enhancement factor ( EF) of about 105 despite of miniaturization.

DNA-Assembled Plasmonic Waveguides for Nanoscale Light Propagation to a Fluorescent Nanodiamond.

Plasmonic waveguides consisting of metal nanoparticle chains can localize and guide light well below the diffraction limit, but high propagation losses due to lithography-limited large interparticle spacing have impeded practical applications. Here, we demonstrate that DNA-origami-based self-assembly of monocrystalline gold nanoparticles allows the interparticle spacing to be decreased to ∼2 nm, thus reducing propagation losses to 0.8 dB per 50 nm at a deep subwavelength confinement of 62 nm (∼λ/10). We characterize the individual waveguides with nanometer-scale resolution by electron energy-loss spectroscopy. Light propagation toward a fluorescent nanodiamond is directly visualized by cathodoluminescence imaging spectroscopy on a single-device level, thereby realizing nanoscale light manipulation and energy conversion. Simulations suggest that longitudinal plasmon modes arising from the narrow gaps are responsible for the efficient waveguiding. With this scalable DNA origami approach, micrometer-long propagation lengths could be achieved, enabling applications in information technology, sensing, and quantum optics.

Design and Synthesis of Triangulated DNA Origami Trusses.

DNA nanotechnology offers unique control over matter on the nanoscale. Here, we extend the DNA origami method to cover a range of wireframe truss structures composed of equilateral triangles, which use less material per volume than standard multiple-helix bundles. From a flat truss design, we folded tetrahedral, octahedral, or irregular dodecahedral trusses by exchanging few connector strands. Other than standard origami designs, the trusses can be folded in low-salt buffers that make them compatible with cell culture buffers. The structures also have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. Our graph routing program and a simple design pipeline will enable other laboratories to make use of this valuable and potent new construction principle for DNA-based nanoengineering.

Block Copolymer Micellization as a Protection Strategy for DNA Origami.

DNA nanotechnology enables the synthesis of nanometer-sized objects that can be site-specifically functionalized with a large variety of materials. For these reasons, DNA-based devices such as DNA origami are being considered for applications in molecular biology and nanomedicine. However, many DNA structures need a higher ionic strength than that of common cell culture buffers or bodily fluids to maintain their integrity and can be degraded quickly by nucleases. To overcome these deficiencies, we coated several different DNA origami structures with a cationic poly(ethylene glycol)-polylysine block copolymer, which electrostatically covered the DNA nanostructures to form DNA origami polyplex micelles (DOPMs). This straightforward, cost-effective, and robust route to protect DNA-based structures could therefore enable applications in biology and nanomedicine where unprotected DNA origami would be degraded.

Bending Unwinds DNA.

In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind DNA, but this effect has not been directly experimentally quantified so far. We developed a ligation-based assay with nicked DNA circles of variable length, thereby decoupling the twist-dependent ligation efficiency from the large bending strain which dominates conventional circularization assays. We demonstrate that tightly bent DNA indeed unwinds to over 11 base pairs/turn, exactly as predicted. Our discovery requires reassessing the molecular mechanisms and energetics of all processes where DNA is tightly bent or relaxed again, including DNA packaging, gene regulation and expression.

Construction of a structurally defined double-stranded DNA catenane.

Topologically interlocked structures like catenanes and rotaxanes are promising components for the construction of molecular machines and motors. Herein we describe the construction of double-stranded DNA catenanes for DNA nanotechnology. For this, C-shaped DNA minicircle fragments were equipped with sequence-specific DNA-binding polyamides and their respective binding site. Formation of catenanes is achieved by self-assembly of two of these fragments and subsequent addition of a ring-closing oligonucleotide.

DNA minicircles connected via G-quadruplex interaction modules.

G-quadruplexes are becoming reliable alternative interaction modules for the construction of DNA nanoarchitectures due to their prompt inducibility by salts. In this Full Paper, we report the design and synthesis of two different DNA minicircles equipped with G-rich appendixes that can self-hybridize into a G-quadruplex, which acts as a DNA recruiter and glue. Both minicircles, one containing a hairpin-like G-rich region and the other an open tuning-fork-like G-rich region, have the potential to form DNA G-nanoconstructs but only the tuning-fork minicircle does so. Incubation of the tuning-fork minicircle with Na(+) and Ni(2+) results in the formation of minicircle dimers, while K(+) and Sr(2+) unexpectedly induce the formation of multimers. Moreover, a catenated DNA nanoconstruct is obtained when the components of the hairpin minicircle are incubated with K(+) or Na(+) and assembled in a stepwise sequence. All nanoconstructs are visualized by atomic force microscopy.

Probing the limits of liquid droplet laser desorption mass spectrometry in the analysis of oligonucleotides and nucleic acids.

In the present work we demonstrate the advantages of LILBID mass spectrometry (laser-induced liquid bead ion desorption) in the analysis of nucleic acids and large oligonucleotides. For established methods like matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), the mass analysis of oligonucleotides or of noncovalent oligonucleotide-protein complexes, in particular of very large ones, still represents a considerable challenge either due to the lack of native solutions or nonspecific adduct formation or due to a reduced salt tolerance or a high charge state of the ions. With LILBID, oligonucleotides, solvated in micro-droplets of aqueous buffer at certain pH and ion strength, are brought into the gas phase by laser ablation. We show that our method is able to detect single- and double-stranded oligonucleotides with high softness, demonstrated by the buffer dependence of the melting of a duplex. The absolute sensitivity is in the attomole range concomitant with a total analyte consumption in the femtomole region. The upper mass limit of oligonucleotides still detected with good signal-to-noise ratio with LILBID is the 1.66 MDa plasmid pUC19. With DNA ladders from short duplexes with sticky ends, we show that LILBID correctly reflects the relative thermodynamic stabilities of the ladders. Moreover, as an example for a specific DNA-protein complex we show that a NF-kappaB p50 homodimer binds sequence specifically to its match DNA. In summary we demonstrate that LILBID, although presently performed only with low mass resolution, due to these advantages, is an alternative mass spectrometric method for the analysis of oligonucleotides in general and of specific noncovalent nucleic acid-protein complexes in particular.

Dynamic DNA nanostructures in biomedicine: Beauty, utility and limits.

DNA composite materials are at the forefront, especially for biomedical science, as they can increase the efficacy and safety of current therapies and drug delivery systems. The specificity and predictability of the Watson-Crick base pairing make DNA an excellent building material for the production of programmable and multifunctional objects. In addition, the principle of nucleic acid hybridization can be applied to realize mobile nanostructures, such as those reflected in DNA walkers that sort and collect cargo on DNA tracks, DNA robots performing tasks within living cells and/or DNA tweezers as ultra-sensitive biosensors. In this review, we present the diversity of dynamic DNA nanostructures functionalized with different biomolecules/functional units, imaging smart biomaterials capable of sensing, interacting, delivery and performing complex tasks within living cells/organisms.

Triangulated Wireframe Structures Assembled Using Single-Stranded DNA Tiles.

The field of structural DNA nanotechnology offers a wide range of design strategies with which to build structures with a desired aspect ratio, size, and shape. Compared with traditional close-packed DNA structures, triangulated wireframe structures require less material per surface or volume unit and improve the stability in biologically relevant conditions due to the reduced electrostatic repulsion. Herein, we expand the design space of the DNA single-stranded tile method to cover a range of anisotropic, finite, triangulated wireframe structures as well as a number of one-dimensional crystalline assemblies. These structures are composed of six-arm junctions with a single double helix as connecting edges that assemble in physiologically relevant salinities. For a reliable folding of the structures, single-stranded spacers 2-4 nucleotides long have to be introduced in the junction connecting neighboring arms. Coarse-grained molecular dynamics simulations using the oxDNA model suggests that the spacers prevent the stacking of DNA helices, thereby facilitating the assembly of planar geometries.

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures.

We have recently constructed a "DNA strut" consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson-Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions.

Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling.

The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

DNA-encircled lipid bilayers.

Lipid bilayers and lipid-associated proteins play crucial roles in biology. As in vivo studies and manipulation are inherently difficult, membrane-mimetic systems are useful for the investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. In this work, we describe a route to leverage the programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs). DEBs are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle, in which up to two alkyl chains per helical turn point to the inside of the toroidal DNA ring. When phospholipids are added, a bilayer is observed to self-assemble within the ring such that the alkyl chains of the oligonucleotides stabilize the hydrophobic rim of the bilayer to prevent formation of vesicles and support thermotropic lipid phase transitions. The DEBs are completely free of protein and can be synthesized from commercially available components using routine equipment. The diameter of DEBs can be varied in a predictable manner. The well-established toolbox from structural DNA nanotechnology, will ultimately enable the rational design of DEBs so that their size, shape or functionalization can be adapted to the specific needs of biophysical investigations of lipidic phases and the properties of membrane proteins embedded into DEB nanoparticle bilayers.

Enhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures.

Cancer stem cells (CSCs) are responsible for drug resistance, tumor recurrence, and metastasis in several cancer types, making their eradication a primary objective in cancer therapy. Glioblastoma Multiforme (GBM) tumors are usually composed of a highly infiltrating CSC subpopulation, which has Nestin as a putative marker. Since the majority of these infiltrating cells are able to elude conventional therapies, we have developed gold nanorods (AuNRs) functionalized with an engineered peptide capable of specific recognition and selective eradication of Nestin positive infiltrating GBM-CSCs. These AuNRs generate heat when irradiated by a near-infrared laser, and cause localized cell damage. Nanoparticle internalization assays performed with GBM-CSCs or Nestin negative cells cultured as two-dimensional (2D) monolayers or embedded in three-dimensional (3D) biodegradable-hydrogels of tunable mechanical properties, revealed that the AuNRs were mainly internalized by GBM-CSCs, and not by Nestin negative cells. The AuNRs were taken up via energy-dependent and caveolae-mediated endocytic mechanisms, and were localized inside endosomes. Photothermal treatments resulted in the selective elimination of GBM-CSCs through cell apoptosis, while Nestin negative cells remained viable. Results also indicated that GBM-CSCs embedded in hydrogels were more resistant to AuNR photothermal treatments than when cultured as 2D monolayers. In summary, the combination of our engineered AuNRs with our tunable hydrogel system has shown the potential to provide an in vitro platform for the evaluation and screening of AuNR-based cancer therapeutics, leading to a substantial advancement in the application of AuNRs for targeted GBM-CSC therapy. STATEMENT OF SIGNIFICANCE: There is an urgent need for reliable and efficient therapies for the treatment of Glioblastoma Multiforme (GBM), which is currently an untreatable brain tumor form with a very poor patient survival rate. GBM tumors are mostly comprised of cancer stem cells (CSCs), which are responsible for tumor reoccurrence and therapy resistance. We have developed gold nanorods functionalized with an engineered peptide capable of selective recognition and eradication of GBM-CSCs via heat generation by nanorods upon NIR irradiation. An in vitro evaluation of nanorod therapeutic activities was performed in 3D synthetic-biodegradable hydrogel models with distinct biomechanical cues, and compared to 2D cultures. Results indicated that cells cultured in 3D were more resistant to photothermolysis than in 2D systems.

Toward Self-Assembled Plasmonic Devices: High-Yield Arrangement of Gold Nanoparticles on DNA Origami Templates.

Plasmonic structures allow the manipulation of light with materials that are smaller than the optical wavelength. Such structures can consist of plasmonically active metal nanoparticles and can be fabricated through scalable bottom-up self-assembly on DNA origami templates. To produce functional devices, the precise and high-yield arrangement of each of the nanoparticles on a structure is of vital importance as the absence of a single particle can destroy the functionality of the entire device. Nevertheless, the parameters influencing the yield of the multistep assembly process are still poorly understood. To overcome this deficiency, we employed a test system consisting of a tubular six-helix bundle DNA origami with binding sites for eight oligonucleotide-functionalized gold nanoparticles. We systematically studied the assembly yield as a function of a wide range of parameters such as ionic strength, stoichiometric ratio, oligonucleotide linker chemistry, and assembly kinetics by an automated high-throughput analysis of electron micrographs of the formed heterocomplexes. Our optimized protocols enable particle placement yields up to 98.7% and promise the reliable production of sophisticated DNA-based multiparticle plasmonic devices for applications in photonics, optoelectronics, and nanomedicine.

Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries.

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.