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Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles. [ABSTRACT FROM AUTHOR]

The switchable reconfiguration of a mixture of two dimers of DNA origami tiles, AB and CD, into a mixture of two DNA origami dimers composed of AD and CB, using a collection of fuel and antifuel strands, is presented. The reversible reconfiguration of the mixture of dimers AB and CD into AD and CB, is followed by labeling each of the tiles with 0, 1, 2, and 3 4× hairpins labels and by imaging the dimer structures by atomic force microscopy. Subjecting the reconfigurable dimer mixtures to a collection of Mg2+-ion-dependent DNAzyme subunits and the substrates consisting of the ROX/BHQ2-modified substrate and the FAM/BHQ1-modified substrate leads to the triggered and programmed switchable operation, in the presence of appropriate fuel and antifuel strands. In the presence of the AB and CD mixture, the DNAzyme subunits cleaving the ROX/BHQ2-modified substrate is switched on, leading to the fluorescence of ROX. The reconfiguration of the AB and CD dimer mixture to the AD and CB dimer mixture leads to the assembly of the DNAzyme subunits that switches on the cleavage of the FAM/BHQ1-modified substrate and to the fluorescence of FAM. By the cyclic and reversible reconfiguration of the AB and CD dimer mixture to the AD and CB dimer mixture, in the presence of the appropriate fuel and antifuel strands, the switchable catalytic operation of two Mg2+-ion-dependent DNAzymes, leading to the fluorescence of ROX or FAM, is demonstrated. [ABSTRACT FROM AUTHOR]

Self-assembled DNA structures enable creation of specific shapes at the nanometer–micrometer scale with molecular resolution. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temperature. An experimentally unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, we use time-resolved small-angle X-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free solution. We show that the DNA device switches from its open to its closed conformation upon addition of MgCl2 in milliseconds, which is close to the theoretical diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concentration-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concentration and molecular alignment. [ABSTRACT FROM AUTHOR]

DNA origami objects allow for accurate positioning of guest molecules in three dimensions. Validation and understanding of design strategies for particle attachment as well as analysis of specific particle arrangements are desirable. Small-angle X-ray scattering (SAXS) is suited to probe distances of nano-objects with subnanometer resolution at physiologically relevant conditions including pH and salt and at varying temperatures. Here, we show that the pair density distribution function (PDDF) obtained from an indirect Fourier transform of SAXS intensities in a model-free way allows to investigate prototypical DNA origami-mediated gold nanoparticle (AuNP) assemblies. We analyze the structure of three AuNP-dimers on a DNA origami block, an AuNP trimer constituted by those dimers, and a helical arrangement of nine AuNPs on a DNA origami cylinder. For the dimers, we compare the model-free PDDF and explicit modeling of the SAXS intensity data by superposition of scattering intensities of the scattering objects. The PDDF of the trimer is verified to be a superposition of its dimeric contributions, that is, here AuNP-DNA origami assemblies were used as test boards underlining the validity of the PDDF analysis beyond pairs of AuNPs. We obtain information about AuNP distances with an uncertainty margin of 1.2 nm. This readout accuracy in turn can be used for high precision placement of AuNP by careful design of the AuNP attachment sites on the DNA-structure and by fine-tuning of the connector types. [ABSTRACT FROM AUTHOR]

We explore the potential of DNA nanotechnology for developing novel optical voltage sensing nanodevices that convert a local change of electric potential into optical signals. As a proof-of-concept of the sensing mechanism, we assembled voltage responsive DNA origami structures labeled with a single pair of FRET dyes. The DNA structures were reversibly immobilized on a nanocapillary tip and underwent controlled structural changes upon application of an electric field. The applied field was monitored through a change in FRET efficiency. By exchanging the position of a single dye, we could tune the voltage sensitivity of our DNA origami structure, demonstrating the flexibility and versatility of our approach. The experimental studies were complemented by coarse-grained simulations that characterized voltage-dependent elastic deformation of the DNA nanostructures and the associated change in the distance between the FRET pair. Our work opens a novel pathway for determining the mechanical properties of DNA origami structures and highlights potential applications of dynamic DNA nanostructures as voltage sensors. [ABSTRACT FROM AUTHOR]

DNA origami is a promising molecular delivery system for a variety of therapeutic applications including cancer therapy, given its capability to fabricate homogeneous nanostructures whose physicochemical properties (size, shape, surface chemistry) can be precisely tailored. However, the correlation between DNA-origami design and internalization efficiency in different cancer cell lines remains elusive. We investigated the cellular uptake of four DNA-origami nanostructures (DONs) with programmed sizes and shapes in multiple human cancer cell lines. The cellular uptake efficiency of DONs was influenced by size, shape, and cell line. Scavenger receptors were responsible for the internalization of DONs into cancer cells. We observed distinct stages of the internalization process of a gold nanoparticle (AuNP)-tagged rod-shape DON, using high-resolution transmission electron microscopy. This study provides detailed understanding of cellular uptake and intracellular trafficking of DONs in cancer cells, and offers new insights for future optimization of DON-based drug delivery systems for cancer treatment. [ABSTRACT FROM AUTHOR]

Fabricating nanocavities in which optically active single quantum emitters are precisely positioned is crucial for building nanophotonic devices. Here we show that self-assembly based on robust DNA-origami constructs can precisely position single molecules laterally within sub-5 nm gaps between plasmonic substrates that support intense optical confinement. By placing single-molecules at the center of a nanocavity, we show modification of the plasmon cavity resonance before and after bleaching the chromophore and obtain enhancements of ≥4 x 10³ with high quantum yield (≥50%). By varying the lateral position of the molecule in the gap, we directly map the spatial profile of the local density of optical states with a resolution of ±1.5 nm. Our approach introduces a straightforward noninvasive way to measure and quantify confined optical modes on the nanoscale. [ABSTRACT FROM AUTHOR]

Three cyclopentadithiophene-difluorophenylene copolymers (named PhF2,3, PhF2,5, and PhF2,6), which differ by the arrangement of fluorines on the phenylene structural unit, were designed and synthesized for the fabrication of organic field-effect transistors (OFETs). Single crystal structures of model compounds representative of the backbone and density functional theory (DFT) were used to estimate the backbone shape for each copolymer. The different substitution arrangements impact the backbone secondary structure through different nonbonding F···H interactions. PhF2,5 and PhF2,6 assumed more linear backbones relative to PhF2,3, which in turn impacts self-assembly and polymer chain alignment on nanogrooved (NG) substrates. A larger improvement of charge carrier mobility for the more linear backbones was achieved when using NG substrates. Among the three polymers, PhF2,6 achieved the highest average field-effect hole mobility (5.1 cm² V-1 s-1). As evidenced by grazing incidence wide-angle X-ray scattering (GIWAXS), thin films of PF2,5 and PF2,6 exhibited a higher degree of anisotropic alignment, relative to the more curved PF2,3 counterpart, consistent with the higher hole mobilities. This work gives insight into how nonbonding interactions can influence charge carrier mobility through changes in secondary structure and suggests that polymers with more linear shapes might be preferred for achieving greater levels of alignment within the confines of a NG environment. [ABSTRACT FROM AUTHOR]

We demonstrate the synthesis of Au nanostar dimers with tunable interparticle gap and controlled stoichiometry assembled on DNA origami. Au nanostars with uniform and sharp tips were immobilized on rectangular DNA origami dimerized structures to create nanoantennas containing monomeric and dimeric Au nanostars. Single Texas red (TR) dye was specifically attached in the junction of the dimerized origami to act as a Raman reporter molecule. The SERS enhancement factors of single TR dye molecules located in the conjunction region in dimer structures having interparticle gaps of 7 and 13 nm are 2 × 1010 and 8 × 109, respectively, which are strong enough for single analyte detection. The highly enhanced electromagnetic field generated by the plasmon coupling between sharp tips and cores of two Au nanostars in the wide conjunction region allows the accommodation and specific detection of large biomolecules. Such DNAdirected assembled nanoantennas with controlled interparticle separation distance and stoichiometry, and well-defined geometry, can be used as excellent substrates in single-molecule SERS spectroscopy and will have potential applications as a reproducible platform in single-molecule sensing. [ABSTRACT FROM AUTHOR]

The dynamics of enzymes are directly associated with their functions in various biological processes. Nevertheless, the ability to image motions of single enzymes in a highly parallel fashion remains a challenge. Here, we develop a DNA origami raft-based platform for in-situ real-time imaging of enzyme cascade at the single-molecule level. The motions of enzymes are rationally controlled via different tethering modes on a two-dimensional (2D) supported lipid bilayer (SLB). We construct an enzyme cascade by anchoring catalase on cholesterol-labeled double-stranded (ds) DNA and glucose oxidase on cholesterol-labeled origami rafts. DNA functionalized with cholesterol can be readily incorporated in SLB via the cholesterol-lipid interaction. By using a total internal reflection fluorescence microscope (TIRFM), we record the moving trajectory of fluorophore-labeled single enzymes on the 2D surface: the downstream catalase diffuses freely in SLB, whereas the upstream glucose oxidase is relatively immobile. By analyzing the trajectories of individual enzymes, we find that the lateral motion of enzymes increases in a substrate concentrationdependent manner and that the enhanced diffusion of enzymes can be transmitted via the cascade reaction. We expect that this platform sheds new light on studying dynamic interactions of proteins and even cellular interactions. [ABSTRACT FROM AUTHOR]

Development of electrically powered DNA origami nanomachines requires effective means to actuate moving origami parts by externally applied electric fields. We demonstrate how origami nanolevers on an electrode can be manipulated (switched) at high frequency by alternating voltages. Orientation switching is long-time stable and can be induced by applying low voltages of 200 mV. The mechanical response time of a 100 nm long origami lever to an applied voltage step is less than 100 μs, allowing dynamic control of the induced motion. Moreover, through voltage assisted capture, origamis can be immobilized from folding solution without purification, even in the presence of excess staple strands. The results establish a way for interfacing and controlling DNA origamis with standard electronics, and enable their use as moving parts in electro-mechanical nanodevices. [ABSTRACT FROM AUTHOR]

In response to environmental variations, living cells need to arrange the conformational changes of macromolecules to achieve the specific biofunctions. Inspired by natural molecular machines, artificial macromolecular assemblies with controllable nanostructures and environmentally responsive functions can be designed. By assembling macromolecular nanostructures with noble metal nanoparticles, environmental information could be significantly amplified and modulated. However, manufacturing dynamic plasmonic nanostructures that are efficiently responsive to different stimuli is still a challenging task. Here we demonstrate a stimulus-responsive plasmonic nanosystem based on DNA origami-organized gold nanorods (GNRs). L-shaped GNR dimers were assembled on rhombus-shaped DNA origami templates. The geometry and chiral signals of the GNR nanoarchitectures respond to multiple stimuli, including glutathione reduction, restriction enzyme action, pH change, or photoirradiation. While the glutathione reduction or restriction enzyme caused irreversible changes in the plasmonic circular dichroism (CD) signals, both pH and light irradiation triggered reversible changes in the plasmonic CD. Our system transduces external stimuli into conformational changes and circular dichroism responses in near-infrared (NIR) wavelengths. By this approach, programmable optical reporters for essential biological signals can be fabricated. [ABSTRACT FROM AUTHOR]

DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems. [ABSTRACT FROM AUTHOR]

DNA is now one of the most widely used molecules for programmed self-assembly of discrete nanostructures. One of the long-standing goals of the DNA nanotechnology field has been the assembly of periodic, macroscopic 3D DNA crystals for controlled positioning of guest molecules to be used in a variety of applications. With continuing successes in assembling DNA crystals, there is an enhanced need to tailor macroscopic crystal propertiesincluding morphologyto enable their integration into more complex systems. Here we describe the ability to alter and control crystal habits of a 3D DNA crystal formed by self-assembly of a DNA 13-mer. The introduction of "poison" oligonucleotides that specifically disrupt critical noncanonical base-pairing interactions in the crystal lattice leads to predictably modified crystal habits. We demonstrate that the poison oligomers can act as habit modifiers both during the initial crystallization and during growth of shell layers on a crystal macroseed. [ABSTRACT FROM AUTHOR]

A split aptamer for adenosine triphosphate (ATP) was embedded as a recognition unit into two levers of a nanomechanical DNA origami construct by extension and modification of selected staple strands. An additional optical module in the stem of the split aptamer comprised two different cyanine-styryl dyes that underwent an energy transfer from green (donor) to red (acceptor) emission if two ATP molecules were bound as target molecule to the recognition module and thereby brought the dyes in close proximity. As a result, the ATP as a target triggered the DNA origami shape transition and yielded a fluorescence color change from green to red as readout. Conventional atomic force microscopy (AFM) images confirmed the topology change from the open form of the DNA origami in the absence of ATP into the closed form in the presence of the target molecule. The obtained closed/open ratios in the absence and presence of target molecules tracked well with the fluorescence color ratios and thereby validated the bicolor fluorescence readout. The correct positioning of the split aptamer as the functional unit farthest away from the fulcrum of the DNA origami was crucial for the aptasensing by fluorescence readout. The fluorescence color change allowed additionally to follow the topology change of the DNA origami aptasensor in real time in solution. The concepts of fluorescence energy transfer for bicolor readout in a split aptamer in solution, and AFM on surfaces, were successfully combined in a single DNA origami construct to obtain a bimodal readout. These results are important for future custom DNA devices for chemical-biological and bioanalytical purposes because they are not only working as simple aptamers but are also visible by AFM on the single-molecule level. [ABSTRACT FROM AUTHOR]

The responses of DNA origami conformation to UV radiation of different wavelengths and doses are investigated. Short- and medium-wavelength UV light can cause photo-lesions in DNA origami. At moderate doses, the lesions do not cause any visible defects in the origami, nor do they significantly affect the hybridization capability. Instead, they help relieve the internal stress in the origami structure and restore it to the designed conformation. At high doses, staple dissociation increases which causes structural disintegration. Long-wavelength UV does not show any effect on origami conformation by itself. We show that this UV range can be used in conjunction with photoactive molecules for photo-reconfiguration, while avoiding any damage to the DNA structures. [ABSTRACT FROM AUTHOR]

An improved method for the metallization of DNA origami is examined in this work. DNA origami, a simple and robust method for creating a wide variety of nanostructured shapes and patterns, provides an enabling template for bottom-up fabrication of next-generation nanodevices. Selective metallization of these DNA templates is needed to make nanoelectronic devices. Here, we demonstrate a metallization process that uses gold nanorod seeds followed by anisotropic plating to provide improved morphology and greater control of the final metallized width of the structure. In our approach, gold nanorods are attached to an origami template to create a seed layer. Electroless gold deposition is then used to fill the gaps between seeds in order to create continuous, conductive nanowires. Importantly, growth during electroless deposition occurs preferentially in the length direction at a rate that is approximately 4 times the growth rate in the width direction, which enables fabrication of narrow, continuous wires. The electrical properties of 49 nanowires with widths ranging from 13 to 29 nm were characterized, and resistivity values as low as 8.9 × 10-7 Ω·m were measured. The anisotropic metallization process presented here represents important progress toward the creation of nanoelectronic devices by molecularly directed placement of functional components onto self-assembled biological templates. [ABSTRACT FROM AUTHOR]

We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies. [ABSTRACT FROM AUTHOR]

The rational design of anisotropic interaction patterns is a key step for programming colloid and nanoparticle self-assembly and emergent functions. Herein, we demonstrate a concept for harnessing the capabilities of 3D DNA origami for extensive supracolloidal self-assembly and showcase its use for making truly monodisperse, patchy, divalent nanocuboids that can self-assemble into supracolloidal fibrils via programmable DNA hybridization. A change in the number of connector duplexes at the patches reveals that multivalency and cooperativity play crucial roles to enhance superstructure formation. We further show thermal and chemical switching of the superstructures as the first steps toward reconfigurable self-assemblies. This concept lays the groundwork for merging monodisperse 3D DNA origami, featuring programmable patchiness and interactions, with nanoparticle self-assembly. [ABSTRACT FROM AUTHOR]

DNA origami nanostructures have been used extensively as scaffolds for numerous applications such as for organizing both organic and inorganic nanomaterials, studying single molecule reactions, and fabricating photonic devices. Yet, little has been done toward the integration of DNA origami nanostructures into nanoelectronic devices. Among other challenges, the technical difficulties in producing well-defined electrical contacts between macroscopic electrodes and individual DNA origami-based nanodevices represent a serious bottleneck that hinders the thorough characterization of such devices. Therefore, in this work, we have developed a method to electrically contact individual DNA origami-based metallic nanowires using electron beam lithography. We then characterize the charge transport of such nanowires in the temperature range from room temperature down to 4.2 K. The room temperature charge transport measurements exhibit ohmic behavior, whereas at lower temperatures, multiple charge transport mechanisms such as tunneling and thermally assisted transport start to dominate. Our results confirm that charge transport along metallized DNA origami nanostructures may deviate from pure metallic behavior due to several factors including partial metallization, seed inhomogeneities, impurities, and weak electronic coupling among AuNPs. Besides, this study further elucidates the importance of variable temperature measurements for determining the dominant charge transport mechanisms for conductive nanostructures made by self-assembly approaches. [ABSTRACT FROM AUTHOR]

Coherent energy exchange between plasmons and excitons is a phenomenon that arises in the strong coupling regime resulting in distinct hybrid states. The DNA-origami technique provides an ideal framework to custom-tune plasmon-exciton nanostructures. By employing this well controlled self-assembly process, we realized hybrid states by precisely positioning metallic nanoparticles in a defined spatial arrangement with fixed nanometer-sized interparticle spacing. Varying the nanoparticle diameter between 30 nm and 60 nm while keeping their separation distance constant allowed us to precisely adjust the plasmon resonance of the structure to accurately match the energy frequency of a J-aggregate exciton. With this system we obtained strong plasmon-exciton coupling and studied far-field scattering at the single-structure level. The individual structures displayed normal mode splitting up to 170 meV. The plasmon tunability and the strong field confinement attained with nanodimers on DNA-origami renders an ideal tool to bottom-up assembly plasmon-exciton systems operating at room temperature. [ABSTRACT FROM AUTHOR]

Self-assembled DNA origami nanostructures enable the creation of precisely defined shapes at the molecular scale. Dynamic DNA devices that are capable of switching between defined conformations could afford completely novel functionalities for diagnostic, therapeutic, or engineering applications. Developing such objects benefits strongly from experimental feedback about conformational changes and 3D structures, ideally in solution, free of potential biases from surface attachment or labeling. Here, we demonstrate that small-angle X-ray scattering (SAXS) can quantitatively resolve the conformational changes of a DNA origami two-state switch device as a function of the ionic strength of the solution. In addition, we show how SAXS data allow for refinement of the predicted idealized three-dimensional structure of the DNA object using a normal mode approach based on an elastic network model. The results reveal deviations from the idealized design geometries that are otherwise difficult to resolve. Our results establish SAXS as a powerful tool to investigate conformational changes and solution structures of DNA origami and we anticipate our methodology to be broadly applicable to increasingly complex DNA and RNA devices. [ABSTRACT FROM AUTHOR]

Scaffolded DNA origami nanostructures enable the self-assembly of arbitrarily shaped objects with unprecedented accuracy. Yet, varying physiological conditions are prone to induce slight structural changes in the nanoscale architecture. Here, we report on high precision measurements of overall shape and interhelical distance of three prototypic DNA origami structures in solution using synchrotron small-angle X-ray scattering. Sheet-, brick-, and cylinder-shaped DNA constructs were assembled and the shape factors determined with angstrom resolution from fits to the scattering profiles. With decreasing MgCl2 concentration electrostatic swelling of both shape cross section and interhelical DNA spacing of the DNA origami structures is observed. The structures tolerate up to 10% interhelical expansion before they disintegrate. In contrast, with increasing temperature, the cylinder-shaped structures show no thermal expansion in a wide temperature window before they abruptly melt above 50 °C. Details on molecular structure of DNA origami can also be obtained using in-house X-ray scattering equipment and, hence, allow for routine folding and stability testing of DNA-based agents that are designed to operate under varying salt conditions. [ABSTRACT FROM AUTHOR]

Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries. [ABSTRACT FROM AUTHOR]

We show hierarchical assembly of plasmonic toroidal metamolecules that exhibit tailored optical activity in the visible spectral range. Each metamolecule consists of four identical origami-templated helical building blocks. Such toroidal metamolecules show a stronger chiroptical response than monomers and dimers of the helical building blocks. Enantiomers of the plasmonic structures yield opposite circular dichroism spectra. Experimental results agree well with the theoretical simulations. We also show that given the circular symmetry of the structures s distinct chiroptical response along their axial orientation can be uncovered via simple spin-coating of the metamolecules on substrates. Our work provides a new strategy to create plasmonic chiral platforms with sophisticated nanoscale architectures for potential applications such as chiral sensing using chemically based assembly systems. [ABSTRACT FROM AUTHOR]

We investigate the kinetics and thermodynamics of DNA origami dimerization using flat rectangle origami components and different architectures of Watson-Crick complementary single-stranded DNA ("sticky end") linking strategies. We systematically vary the number of linkers, the length of the sticky ends on the linker, and linker architecture and measure the corresponding yields as well as forward and reverse reaction rate constants through fluorescence quenching assays. Yields were further verified using atomic force microscopy. We calculate values of H° and ?S° for various interface designs and find nonlinear van't Hoff behavior, best described by two linear equations, suggesting distinct regimes of dimerization between those with and those without well-formed interfaces. We find that self-assembly reactions can be tuned by manipulating the interface architecture without suffering a loss in yield, even when yield is high, ~75-80%. We show that the second-order forward reaction rate constant (kon) depends on both linker architecture and number of linkers used, with typical values on the order of 105-106 (M·s)-1, values that are similar to those of bimolecular association of small, complementary DNA strands. The kon values are generally non-Arrhenius, tending to increase with decreasing temperature. Finally, we use kinetic and thermodynamic information about the optimal linking architecture to extend the system to an infinite, two-component repeating lattice system and show that we can form micron-sized lattices, with well-formed structures up to 8 µm². [ABSTRACT FROM AUTHOR]

DNA circuits tethered to nanoplatforms can perform cascade reactions for signal amplification. One DNA single strand activates a strand-displacement cascade generating numerous outputs, and therefore amplifying the signal. These localized circuits present, however, an important limitation: the spontaneous activation of the cascade reaction. Current methods to stabilize these circuits employ combination of protective DNA strands, which need to be removed to activate the device. This protection-deprotection process generates an important amount of unwanted side reactions. This is indeed an important limitation for the large potential application of these amplification circuits. In the present work, G-quadruplex DNA structures were used to stabilize localized DNA circuits. This new protocol generates nanoplatforms that no longer requires protective-deprotective systems and is therefore completely neutral to the sample. In addition, cations such as Pb2+ or Ca2+ can be also employed to activate the device enlarging the potential applications from biosensors devices to metal detector sensors. [ABSTRACT FROM AUTHOR]

DNA nanoassemblies, such as DNA origamis, hold promise in biosensing, drug delivery, nanoelectronic circuits, and biological computing, which require suitable methods for migration and precision positioning. Insulator-based dielectrophoresis (iDEP) has been demonstrated as a powerful migration and trapping tool for µm- and nm-sized colloids as well as DNA origamis. However, little is known about the polarizability of origami species, which is responsible for their dielectrophoretic migration. Here, we report the experimentally determined polarizabilities of the six-helix bundle origami (6HxB) and triangle origami by measuring the migration times through a potential landscape exhibiting dielectrophoretic barriers. The resulting migration times correlate to the depth of the dielectrophoretic potential barrier and the escape characteristics of the origami according to an adapted Kramer's rate model, allowing their polarizabilities to be determined. We found that the 6HxB polarizability is larger than that of the triangle origami, which correlates with the variations in charge density of both origamis. Further, we discuss the orientation of both origami species in the dielectrophoretic trap and discuss the influence of diffusion during the escape process. Our study provides detailed insight into the factors contributing to the migration through dielectrophoretic potential landscapes, which can be exploited for applications with DNA and other nanoassemblies based on dielectrophoresis. [ABSTRACT FROM AUTHOR]

Deterministic placement and dynamic manipulation of individual plasmonic nanoparticles with nanoscale precision feature an important step toward active nanoplasmonic devices with prescribed levels of performance and functionalities at optical frequencies. In this Letter, we demonstrate a plasmonic walker couple system, in which two gold nanorod walkers can independently or simultaneously perform stepwise walking powered by DNA hybridization along the same DNA origami track. We utilize optical spectroscopy to resolve such dynamic walking with nanoscale steps well below the optical diffraction limit. We also show that the number of walkers and the optical response of the system can be correlated. Our studies exemplify the power of plasmonics, when integrated with DNA nanotechnology for realization of advanced artificial nanomachinery with tailored optical functionalities. [ABSTRACT FROM AUTHOR]

Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot. [ABSTRACT FROM AUTHOR]

DNAorigami has become an established technique for designing well-definednanostructures with any desired shape and for the controlled arrangementof functional nanostructures with few nanometer resolution. Theseunique features make DNA origami nanostructures promising candidatesfor use as scaffolds in nanoelectronics and nanophotonics device fabrication.Consequently, a number of studies have shown the precise organizationof metallic nanoparticles on various DNA origami shapes. In this work,we fabricated large arrays of aligned DNA origami decorated with ahigh density of gold nanoparticles (AuNPs). To this end, we firstdemonstrate the high-yield assembly of high-density AuNP arrangementson DNA origami adsorbed to Si surfaces with few unbound backgroundnanoparticles by carefully controlling the concentrations of MgCl2and AuNPs in the hybridization buffer and the hybridizationtime. Then, we evaluate two methods, i.e., hybridization to prealignedDNA origami and molecular combing in a receding meniscus, with respectto their potential to yield large arrays of aligned AuNP-decoratedDNA origami nanotubes. Because of the comparatively low MgCl2concentration required for the efficient immobilization of the AuNPs,the prealigned DNA origami become mobile and displaced from theiroriginal positions, thereby decreasing the alignment yield. This increasedmobility, on the other hand, makes the adsorbed origami susceptibleto molecular combing, and a total alignment yield of 86% is obtainedin this way. [ABSTRACT FROM AUTHOR]

Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA’s non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms. [ABSTRACT FROM AUTHOR]

Availability of basic tools for engineering molecular systems with precisely defined properties is crucial toward progress in development of new responsive materials. Among such materials are systems capable of generating an ultrasensitive response (i.e., large relative changes in output in response to small changes in input). Herein, we focus on a rational design of DNA quadruplex based structures as ultrasensitive response elements. In particular, we demonstrate how addition of allosteric guiding elements can be engineered into H+-responsive i-motif structure to yield maximized response sensitivity. [ABSTRACT FROM AUTHOR]

As a step toward deterministic and scalable assembly of ordered spin arrays we here demonstrate a bottom-up approach to position fluorescent nanodiamonds (NDs) with nanometer precision on DNA origami structures. We have realized a reliable and broadly applicable surface modification strategy that results in DNA-functionalized and perfectly dispersed NDs that were then self-assembled in predefined geometries. With optical studies we show that the fluorescence properties of the nitrogen-vacancy color centers in NDs are preserved during surface modification and DNA assembly. As this method allows the nanoscale arrangement of fluorescent NDs together with other optically active components in complex geometries, applications based on self-assembled spin lattices or plasmon-enhanced spin sensors as well as improved fluorescent labeling for bioimaging could be envisioned. [ABSTRACT FROM AUTHOR]

Scaffolded DNA origami enables thefabrication of a variety of complex nanostructures that promise utilityin diverse fields of application, ranging from biosensing over advancedtherapeutics to metamaterials. The broad applicability of DNA origamias a material beyond the level of proof-of-concept studies criticallydepends, among other factors, on the availability of large amountsof pure single-stranded scaffold DNA. Here, we present a method forthe efficient production of M13 bacteriophage-derived genomic DNAusing high-cell-density fermentation of Escherichia coliin stirred-tank bioreactors. We achieve phage titers of up to 1.6× 1014plaque-forming units per mL. Downstream processingyields up to 410 mg of high-quality single-stranded DNA per one literreaction volume, thus upgrading DNA origami-based nanotechnology fromthe milligram to the gram scale. [ABSTRACT FROM AUTHOR]

Fastand efficient transport of molecular cargoes along tracks or on supramolecularplatforms is an important prerequisite for the development of futurenanorobotic systems and assembly lines. Here, we study the diffusivetransport of DNA cargo strands bound to a supramolecular DNA origamistructure via an extended tether arm. For short distances (on theorder of a few nanometers), transport from a start to a target siteis found to be less efficient than for direct transfer without tether.For distances on the scale of the origami platform itself, however,cargo transfer mediated by a rigid tether arm occurs very fast androbust, whereas a more flexible, hinged tether is found to be considerablyless efficient. Our results suggest diffusive motion on a moleculartether as a highly efficient mechanism for fast transfer of cargoesover long distances. [ABSTRACT FROM AUTHOR]

Structural DNA nanotechnology provides a feasible technique for the design and fabrication of complex geometries even exhibiting controllable dynamic behavior. Recently we have demonstrated the possibility of implementing macroscopic engineering design approaches to construct DNA origami mechanisms (DOM) with programmable motion and tunable flexibility. Here, we implement the design of compliant DNA origami mechanisms to extend from prescribing motion to prescribing an energy landscape. Compliant mechanisms facilitate motion via deformation of components with tunable stiffness resulting in well-defined mechanical energy stored in the structure. We design, fabricate, and characterize a DNA origami nanostructure with an energy landscape defined by two stable states (local energy minima) separated by a designed energy barrier. This nanostructure is a four-bar bistable mechanism with two undeformed states. Traversing between those states requires deformation, and hence mechanical energy storage, in a compliant arm of the linkage. The energy barrier for switching between two states was obtained from the conformational distribution based on a Boltzmann probability function and closely follows a predictive mechanical model. Furthermore, we demonstrated the ability to actuate the mechanism into one stable state via additional DNA inputs and then release the actuation via DNA strand displacement. This controllable multistate system establishes a foundation for direct design of energy landscapes that regulate conformational dynamics similar to biomolecular complexes. [ABSTRACT FROM AUTHOR]

Ionized nucleobases participate in pairing interactions outside of Watson and Crick's rules. Base pairing and ionization can be coupled via global conformational changes to raise the apparent pKa of protonated nucleobases to values above physiological pH. To provide the first specific reporter of proton-coupled DNA folding, we developed a "push--pull" fluorescent nucleoside analog composed of dimethylaniline (DMA) fused to deoxycytidine. "DMAC" exhibits the same pKa and base pairing characteristics as native cytosine residues in the human telomeric repeat sequence, where it causes little or no perturbation of DNA structure or stability. Upon protonation of DMAC, enhanced charge transfer results in large red shifting (+40 nm) of its excitation/emission maxima. DMAC's fluorescence intensity, anisotropy, and energy transfer properties can be used to track conformational changes in real time. Strand displacement assays were conducted by mixing DMAC-labeled duplexes containing a 5' single-stranded overhang with an excess of unlabeled DNA to initiate thermodynamically favorable unfolding--refolding reactions that release the DMAC-labeled strand from its complement. Rate constants for strand displacement upon addition of i-motif DNA (k = 1.0 M-1 s-1, t1/2 ≈ 12 h) were 320-fold lower than those measured upon addition of unfolded DNA (k = 3.2 x 10² M-1 s-1, t1/2 ≈ 2 min). These results reveal that i-motif structures having only marginal thermodynamic stabilities (Tm < 40 °C) can still pose large kinetic barriers to duplex formation under near-physiological conditions of pH (5.75), temperature (25 °C), and salt (100 mM NaCl). [ABSTRACT FROM AUTHOR]

While understanding translocationof DNA through a solid-state nanopore is vital for exploiting itspotential for sensing and sequencing at the single-molecule level,surprisingly little is known about the dynamics of the propagationof DNA through the nanopore. Here we use linear double-stranded DNAmolecules, assembled by the DNA origami technique, with markers atknown positions in order to determine for the first time the localvelocity of different segments along the length of the molecule. Weobserve large intramolecular velocity fluctuations, likely relatedto changes in the drag force as the DNA blob unfolds. Furthermore,we observe an increase in the local translocation velocity towardthe end of the translocation process, consistent with a speeding updue to unfolding of the last part of the DNA blob. We use the velocityprofile to estimate the uncertainty in determining the position ofa feature along the DNA given its temporal location and demonstratethe error introduced by assuming a constant translocation velocity. [ABSTRACT FROM AUTHOR]

We introducea simple experimental system to study dynamics of needle-like nanoobjectsin two dimensions (2D) as a function of their surface density closeto the isotropic–nematic transition. Using fluorescence correlationspectroscopy, we find that translational and rotational diffusionof rigid DNA origami nanoneedles bound to freestanding lipid membranesis strongly suppressed upon an increase in the surface particle density.Our experimental observations show a good agreement with results ofMonte Carlo simulations of Brownian hard needles in 2D. [ABSTRACT FROM AUTHOR]

We introduce a new concept that allowsthe synthesis of inorganicnanoparticles with programmable shape. Three-dimensional DNA origaminanostructures harboring an internal cavity are used as molds. A smallgold nanoparticle within the cavity nucleates solution-based golddeposition leading to mold filling. We demonstrate the fabricationof 40 nm long rodlike gold particles with quadratic cross sectionand the formation of higher order assemblies of the obtained particles,which is mediated by their DNA shell. [ABSTRACT FROM AUTHOR]

StructuralDNA nanotechnology, and specifically scaffolded DNA origami, is rapidlydeveloping as a versatile method for bottom-up fabrication of novelnanometer-scale materials and devices. However, lengths of conventionalsingle-stranded scaffolds, for example, 7,249-nucleotide circulargenomic DNA from the M13mp18 phage, limit the scales of these uniquelyaddressable structures. Additionally, increasing DNA origami sizegenerates the cost burden of increased staple-strand synthesis. Weaddressed this 2-fold problem by developing the following methods:(1) production of the largest to-date biologically derived single-strandedscaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotideDNA in a circular, single-stranded form and (2) inexpensive DNA synthesisvia an inkjet-printing process on a chip embossed with functionalizedmicropillars made from cyclic olefin copolymer. We have experimentallydemonstrated very efficient assembly of a 51-kilobasepair origamifrom the λ/M13 hybrid scaffold folded by chip-derived staplestrands. In addition, we have demonstrated two-dimensional, asymmetricorigami sheets with controlled global curvature such that they landon a substrate in predictable orientations that have been verifiedby atomic force microscopy. [ABSTRACT FROM AUTHOR]

Wereport the effect of chemical and physical treatments on thestructural stability of DNA origami nanostructures. Our result showsthat DNA nanostructure maintains its shape under harsh processingconditions, including thermal annealing up to 200 °C for 10 min,immersing in a wide range of organic solvents for up to 24 h, briefexposure to alkaline aqueous solutions, and 5 min exposure to UV/O3. Our result suggests that the application window of DNA nanostructureis significantly wider than previously believed. [ABSTRACT FROM AUTHOR]

Thedependence of quantum dot (QD) fluorescence emission on theproximity of 30 nm gold nanoparticles (AuNPs) was studied with controlledinterparticle distances ranging from 15 to 70 nm. This was achievedby coassembling DNA-conjugated QDs and AuNPs in a 1:1 ratio at precisepositions on a triangular-shaped DNA origami platform. A profound,long-range quenching of the photoluminescence intensity of the QDswas observed. A combination of static and time-resolved fluorescencemeasurements suggests that the quenching is due to an increase inthe nonradiative decay rate of QD emission. Unlike FRET, the energytransfer is inversely proportional to the 2.7th power of the distancebetween nanoparticles with half quenching at ∼28 nm. This long-rangequenching phenomena may be useful for developing extended spectroscopicrulers in the future. [ABSTRACT FROM AUTHOR]

We report the effect of the length and identity of a nontarget binding spacer in both the probe and target sequences on the overall performance of a folding-based electrochemical DNA sensor. Six near-identical DNA probes were used in this study; the main differences between these probes are the length (6, 10, or 14 bases) and identity (thymine (T) or adenine (A)) of the spacer connecting the two target binding domains. Despite the differences, the signaling mechanism of these sensors remains essentially the same. The methylene blue (MB)-modified probe assumes a linear unstructured conformation in the absence of the target; upon hybridization to the target, the probe adopts a "close" conformation, resulting in an increase in the MB current. Among the six sensors, the T14 and A14 sensors showed the largest signal increase upon target hybridization, highlighting the significance of probe flexibility on sensor performance. In addition to the target without a midsequence spacer, 12 other targets, each with a different oligo-T or oligo-A spacer, were used to elucidate the effect of target flexibility on the sensors' signaling capacity. For all six sensors, hybridization to targets with a 2- or 3-base spacer resulted in the largest signal increase. Higher signal enhancement was also observed with targets with an oligo-A spacer. For this sensor design, addition of a long nontarget binding spacer to the probe sequence is advantageous, as it provides flexibility for optimal target capture. The length of the spacer in the target sequence, however, should be adequately long to enable efficient hybridization yet does not introduce undesirable electrostatic and crowding effects. [ABSTRACT FROM AUTHOR]

Over the past three decades DNA has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular Watson-Crick base-pairing interactions. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures of increasing complexity. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, DNA computing, nanorobotics, disease diagnosis, and drug delivery. This Perspective discusses the state of the art in the field of structural DNA nanotechnology and presents some of the challenges and opportunities that exist in DNA-based molecular design and programming. [ABSTRACT FROM AUTHOR]

Nucleicacids have become powerful building blocks for creatingsupramolecular nanostructures with a variety of new and interestingbehaviors. The predictable and guided folding of DNA, inspired bynature, allows designs to manipulate molecular-scale processes unlikeany other material system. Thus, DNA can be co-opted for engineeredand purposeful ends. [ABSTRACT FROM AUTHOR]

DNA based nanotechnology provides a basis forhigh-resolution fabricationof objects almost without physical size limitations. However, thepathway to large-scale production of large objects is currently unclear.Operationally, one method forward is to use high information content,large building blocks, which can be generated with high yield andreproducibility. Although flat DNA origami naturally invites comparisonto pixels in zero, one, and two dimensions and voxels in three dimensionsand has provided an excellent mechanism for generating blocks of significantsize and complexity and a multitude of shapes, the field is youngenough that a single “brick” has not become the standardplatform used by the majority of researchers in the field. [ABSTRACT FROM AUTHOR]

Eight years have passed since the striking debut of the DNA origamitechnique (Rothemund, P. W. K. Nature2006, 440, 297−302), in which long single-strandedDNA is folded into a designed nanostructure, in either 2D or 3D, withthe aid of many short staple strands. The number of proposals fornew design principles for DNA origami structures seems to have alreadyreached a peak. It is apparent that DNA origami study is now enteringthe second phase of creating practical applications. The developmentof functional nanomechanical molecular devices using the DNA origamitechnique is one such application attracting significant interestfrom researchers in the field. Nanomechanical DNA origami devices,which maintain the characteristics of DNA origami structures, havevarious advantages over conventional DNA nanomachines. Comparativelyhigh assembly yield, relatively large size visible via atomic forcemicroscopy (AFM) or transmission electron microscopy (TEM), and thecapability to assemble multiple functional groups with precision usingmultiple staple strands are some of the advantages of the DNA origamitechnique for constructing sophisticated molecular devices. [ABSTRACT FROM AUTHOR]

Self-assembled functional DNA oligonucleotidebased architecturesrepresent highly promising candidates for the creation of nanoscaledevices. The field of DNA nanotechnology has emerged to a high levelof maturity and currently constitutes one of the most dynamic, creative,and exciting modern research areas. The transformation from structuralDNA nanotechnology to functional DNA architectures is already takingplace with tremendous pace. Particularly the advent of DNA origamitechnology has propelled DNA nanotechnology forward. DNA origami provideda versatile method for precisely aligning structural and functionalDNA modules in two and three dimensions, thereby serving as a meansfor constructing scaffolds and chassis required for the precise orchestrationof multiple functional DNA architectures. Key modules of these willcontain interlocked nanomechanical components made of DNA. The mechanicalinterlocking allows for performing highly specific and controlledmotion, by reducing the dimensionality of diffusion-controlled processeswithout restrictions in motional flexibility. [ABSTRACT FROM AUTHOR]