A Bipedal DNA Motor that Travels Back and Forth between Two DNA Origami Tiles

In this work, the successful operation of a dynamic DNA device constructed from two DNA origami building blocks is reported. The device includes a bipedal walker that strides back and forth between the two origami tiles. Two different DNA origami tiles are first prepared separately; they are then joined together in a controlled manner by a set of DNA strands to form a stable track in high yield as confirmed by single‐molecule fluorescence (SMF). Second, a bipedal DNA motor, initially attached to one of the two origami units and operated by sequential interaction with “fuel” and “antifuel” DNA strands, moves from one origami tile to another and then back again. The operational yield, measured by SMF, was similar to that of a motor operating on a similar track embedded in a single origami tile, confirming that the transfer across the junction from one tile to the other does not result in dissociation that is any more than that of steps on a single tile. These results demonstrate that moving parts can reliably travel from one origami unit to another, and it demonstrates the feasibility of dynamic DNA molecular machines that are made of more than a single origami building block. This study is a step toward the development of motors that can stride over micrometer distances.     

Programming Motions of DNA Origami Nanomachines

DNA nanotechnology enables the precise fabrication of DNA‐based machines with nanoscale dimensions. A wide range of DNA nanomachines are designed, which can be activated by specific inputs to perform various movement and functions. The excellent rigidity and unprecedented addressability of DNA origami have made it an excellent platform for manipulating and investigating the motion behaviors of DNA machines at single‐molecule level. In this Concept, power supply, machine actuation, and motion behavior of DNA machines on origami platforms are summarized and classified. The strategies utilized for programming motion behavior of DNA machines on DNA origami are also discussed with representative examples. The challenges and outlook for future development of manipulating DNA nanomachines at the single molecule level are presented and discussed.     

Counting Fluorescent Dye Molecules on DNA Origami by Means of Photon Statistics

Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single‐molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1–20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well‐defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non‐invasively within 150 ms. The method might become a quantifying add‐on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT‐type microscopy.     

Self‐Assembly of DNA Origami Heterodimers in High Yields and Analysis of the Involved Mechanisms

Efficient fabrication of structurally and functionally diverse nanomolecular devices and machines by organizing separately prepared DNA origami building blocks into a larger structure is limited by origami attachment yields. A general method that enables attachment of origami building blocks using ‘sticky ends' at very high yields is demonstrated. Two different rectangular origami monomers are purified using agarose gel electrophoresis conducted in solute containing 100 × 10^?3 m NaCl, a treatment that facilitates the dissociation of most of the incorrectly hybridized origami structures that form through blunt‐end interactions during the thermal annealing process and removes these structures as well as excess strands that otherwise interfere with the desired heterodimerization reaction. Heterodimerization yields of gel‐purified monomers are between 98.6% and 99.6%, considerably higher than that of monomers purified using the polyethylene glycol (PEG) method (88.7–96.7%). Depending on the number of PEG purification rounds, these results correspond to about 4‐ to 25‐fold reduction in the number of incorrect structures observed by atomic force microscopy. Furthermore, the analyses of the incorrect structures observed before and after the heterodimerization reactions and comparison of the purification methods provide valuable information on the reaction mechanisms that interfere with heterodimerization.     

Cation‐Induced Stabilization and Denaturation of DNA Origami Nanostructures in Urea and Guanidinium Chloride

The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and single‐molecule biophysics. Here, the effect of Na⁺ and Mg²⁺ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting‐out of Gdm⁺ to the hydrophobic DNA base stack. The effect of cation‐induced DNA origami denaturation by GdmCl deserves consideration in the design of single‐molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes.     

Rhombic‐Shaped Nanostructures and Mechanical Properties of 2D DNA Origami Constructed with Different Crossover/Nick Designs

DNA origami methods enable the fabrication of various nanostructures and nanodevices, but their effective use depends on an understanding of their structural and mechanical properties and the effects of basic structural features. Frequency‐modulation atomic force microscopy is introduced to directly characterize, in aqueous solution, the crossover regions of sets of 2D DNA origami based on different crossover/nick designs. Rhombic‐shaped nanostructures formed under the influence of flexible crossovers placed between DNA helices are observed in DNA origami incorporating crossovers every 3, 4, or 6 DNA turns. The bending rigidity of crossovers is determined to be only one‐third of that of the DNA helix, based on interhelical electrostatic forces reported elsewhere, and the measured pitches of the 3‐turn crossover design rhombic‐shaped nanostructures undergoing negligible bending. To evaluate the robustness of their structural integrity, they are intentionally and simultaneously stressed using force‐controlled atomic force microscopy. DNA crossovers are verified to have a stabilizing effect on the structural robustness, while the nicks have an opposite effect. The structural and mechanical properties of DNA origami and the effects of crossovers and nicks revealed in this paper can provide information essential for the design of versatile DNA origami structures that exhibit specified and desirable properties.     

Co‐Immobilization of Proteins and DNA Origami Nanoplates to Produce High‐Contrast Biomolecular Nanoarrays

The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co‐immobilize proteins with DNA origami at pre‐determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co‐binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm‐wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read‐out. The co‐immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami‐protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single‐molecule protein arrays.     

The Brownian and Flow‐Driven Rotational Dynamics of a Multicomponent DNA Origami‐Based Rotor

Nanomechanical devices are becoming increasingly popular due to the very diverse field of potential applications, including nanocomputing, robotics, and drug delivery. DNA is one of the most promising building materials to realize complex 3D structures at the nanoscale level. Several mechanical DNA origami structures have already been designed capable of simple operations such as a DNA box with a controllable lid, bipedal walkers, and cargo sorting robots. However, the nanomechanical properties of mechanically interlinked DNA nanostructures that are in general highly deformable have yet to be extensively experimentally evaluated. In this work, a multicomponent DNA origami‐based rotor is created and fully characterized by electron microscopy under negative stain and cryo preparations. The nanodevice is further immobilized on a microfluidic chamber and its Brownian and flow‐driven rotational behaviors are analyzed in real time by single‐molecule fluorescence microscopy. The rotation in previous DNA rotors based either on strand displacement, electric field or Brownian motion. This study is the first to attempt to manipulate the dynamics of an artificial nanodevice with fluidic flow as a natural force.     

A DNA Origami Mechanical Device for the Regulation of Microcosmic Structural Rigidity

DNA origami makes it feasible to fabricate a tremendous number of DNA nanostructures with various geometries, dimensions, and functionalities. Moreover, an increasing amount of research on DNA nanostructures is focused on biological and biomedical applications. Here, the reversible regulation of microcosmic structural rigidity is accomplished using a DNA origami device in vitro. The designed DNA origami monomer is composed of an internal central axis and an external sliding tube. Due to the external tube sliding, the device transforms between flexible and rigid states. By transporting the device into the liposome, the conformational change of the origami device induces a structural change in the liposome. The results obtained demonstrate that the programmed DNA origami device can be applied to regulate the microcosmic structural rigidity of liposomes. Because microcosmic structural rigidity is important to cell proliferation and function, the results obtained potentially provide a foundation for the regulation of cell microcosmic structural rigidity using DNA nanostructures.     

Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label

The hepatitis B virus (HBV) genotyping may profoundly affect the accurate diagnosis and antiviral treatment of viral hepatitis. Existing genotyping methods such as serological, immunological, or molecular testing are still suffered from substandard specificity and low sensitivity in laboratory or clinical application. In a previous study, a set of high‐efficiency hybridizable DNA origami‐based shape ID probes to target the templates through which genetic variation could be determined in an ultrahigh resolution of atomic force microscopy (AFM) nanomechanical imaging are established. Here, as a further confirmatory research to explore the sensitivity and applicability of this assay, differentially predesigned DNA origami shape ID probes are also developed for precisely HBV genotyping. Through the specific identification of visualized DNA origami nanostructure with clinical HBV DNA samples, the genetic variation information of genotypes can be directly identified under AFM. As a proof‐of‐concept, five genotype B and six genotype C are detected in 11 HBV‐infected patients' blood DNA samples of Han Chinese population in the single‐blinded test. The AFM image‐based DNA origami shape ID genotyping approach shows high specificity and sensitivity, which could be promising for virus infection diagnosis and precision medicine in the future.     

Alignment and Graphene‐Assisted Decoration of Lyotropic Chromonic Liquid Crystals Containing DNA Origami Nanostructures

Composites of DNA origami nanostructures dispersed in a lyotropic chromonic liquid crystal are studied by polarizing optical microscopy. The homogeneous aqueous dispersions can be uniformly aligned by confinement between two glass substrates, either parallel to the substrates owing to uniaxial rubbing or perpendicular to the substrates using ozonized graphene layers. These opportunities of uniform alignment may pave the way for tailored anisometric plasmonic DNA nanostructures to photonic materials. In addition, a decorated texture with nonuniform orientation is observed on substrates coated with pristine graphene. When the water is allowed to evaporate slowly, microscopic crystal needles appear, which are aligned along the local orientation of the director. This decoration method can be used for studying the local orientational order and the defects in chromonic liquid crystals.     

Structural Properties and Surface Modification Decided Pharmacokinetic Behavior and Bio-Distribution of DNA Origami Frameworks in Mice

This study establishes and validates a series of three dimentional (3D) DNA origami frameworks (DOFs) carrying imaging probes to evaluate their pharmacokinetics and real-time bio-distribution in mice. Three typical DOFs with distinguished structural properties are subjected to mice intravenous injection to systematically investigate their in vivo behaviors. Tracing the radioisotope zirconium-89 (⁸⁹Zr) trapped at the inner space of the frameworks, positron emission tomography (PET) imaging is employed to record the real-time bio-distribution of the structures and acquire their pharmacokinetic parameters in the major metabolic organs. The 3D DOFs show different behavior compared to previous structures, with lower kidney accumulation and higher liver retention. Modifications to the structures, such as exposed ssDNA or polyethylene glycol (PEG) moieties, impact their behavior, but are structure-dependent. The 43 nm icosahedra framework among the DOFs perform the best in liver targeting, with the ssDNA extensions enhancing this tendency. The modification of triantennary N-acetylgalactosamine (GalNAc), further improves its uptake in liver cells, especially in hepatocytes over other cell types, discovered by flow cytometry analysis.     

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin‐Receptor Internalization Pathway

DNA origami provides rapid access to easily functionalized, nanometer‐sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA‐based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA‐directed, site‐selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22‐fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.     

Perspectives and Challenges of Emerging Single‐Molecule DNA Sequencing Technologies

The growing demand for analysis of the genomes of many species and cancers, for understanding the role of genetic variation among individuals in disease, and with the ultimate goal of deciphering individual human genomes has led to the development of non‐Sanger reaction‐based technologies towards rapid and inexpensive DNA sequencing. Recent advancements in new DNA sequencing technologies are changing the scientific horizon by dramatically accelerating biological and biomedical research and promising an era of personalized medicine for improved human health. Two single‐molecule sequencing technologies based on fluorescence detection are already in a working state. The newly launched and emerging single‐molecule DNA sequencing approaches are reviewed here. The current challenges of these technologies and potential methods of overcoming these challenges are critically discussed. Further research and development of single‐molecule sequencing will allow researchers to gather nearly error‐free genomic data in a timely and inexpensive manner.

     

Controlling the formation of DNA origami structures with external signals

Degradable Newkome-type and polylysine dendrons functionalized with spermine surface units are used to control the formation of DNA origami structures. The intact dendrons form polyelectrolyte complexes with the scaffold strands, therefore blocking the origami formation. Degradation of the dendron with an optical trigger or chemical reduction leads to the release of the DNA scaffold and efficient formation of the desired origami structure. These results provide new insights towards realizing responsive materials with DNA origami.