Although Wingless (Wg)/Wnt signaling has been implicated in heart development of multiple organisms, conflicting results have been reported regarding the role of Wnt/␤-catenin pathway in cardiac myogenesis: Wg/armadillo signaling promotes heart development in Drosophila, whereas activation of Wnt/␤-catenin signaling inhibits heart formation in avians and amphibians. Using an in vitro system of mouse ES cell differentiation into cardiomyocytes, we show here that Wnt/␤-catenin signaling exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis. Activation of the Wnt/␤-catenin pathway in the early phase during embryoid body (EB) formation enhances ES cell differentiation into cardiomyocytes while suppressing the differentiation into hematopoietic and vascular cell lineages. In contrast, activation of Wnt/␤-catenin signaling in the late phase after EB formation inhibits cardiomyocyte differentiation and enhances the expression of hematopoietic/vascular marker genes through suppression of bone morphogenetic protein signaling. Thus, Wnt/␤-catenin signaling exhibits biphasic and antagonistic effects on cardiomyogenesis and hematopoiesis/vasculogenesis, depending on the stage of development.cardiogenesis

Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor 1-12 and fuel [13][14][15] , all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold 16 , and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.An effective linear molecular motor must traverse its track without dissociating [1][2][3][4][5][6][7]10,12 and run unidirectionally without external intervention [4][5][6][7][8][9][10][11][12] . Directionality may be imposed by the sequential addition of DNA instructions 1-3 or, for autonomous motors, by modifying the track sites that have been visited 5,6,12 , by coupling motion to a unidirectional reaction cycle 4,9,12 or by coordinating the conformation changes of different parts of the motor 11,12 . DNA motors that satisfy all these criteria have typically been demonstrated on tracks that allow only 1-3 steps, although a stochastic DNA 'spider' with many legs has been shown to move longer distances by biased diffusion 17 along a 100 nm track 18 .We have investigated the motion of a simple directional and processive motor fuelled by DNA hydrolysis 6 along an extended track consisting of a one-dimensional array of single-stranded attachment sites (stators), separated by 6 nm. An extended track of 15 identical stators, flanked with special start and stop stators 6 , was assembled on a rectangular DNA origami tile measuring 100 nm × 70 nm (ref. 16; Fig. 1, Supplementary Figs S1, S2). The tile comprises a 7,249-nucleotide (nt) single-stranded DNA template (genome of bacteriophage M13) hybridized to short synthetic staple strands such that the final tile consists of a raft of 24 parallel double helices tethered by the crossover of staples. Two tile designs were used. The helices of tile type A are crosslinked at 16 bp intervals, creating slight underwinding (10.7 bp per turn), which is expected to lead to a global right-handed twisting of the tile 19 . Tile type B is designed to reduce this twist: the average distance between crossovers is 15.6 bp (giving 10.4 bp per turn). The centre and ends of each staple are positioned on opposite surfaces of the tile. Selected staples were extended to include either the 22-nt stator sequence at the 5 ′ end or a hairpin at the centre 16 (Fig. 1a). Stators hybridized to complementary motor strands are visible in atomic force microscope (AFM) images...

Synthetic molecular motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been observed on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks containing four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, molecular systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure.

We herein report the real-time observation of G-quadruplex formation by monitoring the G-quadruplex-induced global change of two duplexes incorporated in a DNA nanoscaffold. The introduced G-rich strands formed an interstrand (3 + 1) G-quadruplex structure in the presence of K(+), and the formed four-stranded structure was disrupted by removal of K(+). These conformational changes were visualized in a nanoscaffold in real-time with fast-scanning atomic force microscopy.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.

T he unique structural motifs and molecular recognition properties of DNA make it a promising template for building nanostructures. 1Ϫ4 Using a long single-stranded DNA as a template, a novel strategy, the so-called DNA origami method, has been developed for the preparation of various two-dimensional (2D) and three-dimensional (3D) nanostructures with defined size. 5Ϫ12 In addition, these origami nanostructures have been used as a platform for the nanopatterning of proteins, nanoparticles, transition metals, and other functional components into deliberately designed arrangements. 13Ϫ20 They can also act as templates for the growth of nanowires, aid in the structural determination of proteins, and provide new platforms for genomic applications. 21 However, these structures offer a relatively small area, which is not sufficient for the precise positioning of functional molecules, and a larger assembly with a size of a few micrometers is required for the preparation of practical devices. For instance, conventional photolithography techniques require a size domain of 1 m. Although programmed DNA and RNA assemblies with the size of around 10Ϫ20 nm have been achieved, 22Ϫ24 strategies for the construction of defined larger assemblies are limited.The size of the origami was first expanded by Rothemund with the assembly of triangular origami into a hexagon with a yield of Ͻ2%. 5 Later, 3D heterotrimers in the shape of a wireframe icosahedron were reported with no information about the yield of the assembled structures. 10 We have recently developed a new method to scale up DNA origami using jigsaw pieces (JPs) and successfully prepared a unidirectional DNA assembly. 25 However, the 2D construction of origami tiles is critical for the development of DNA origami technology. The 2D scale-up of the origami structure was recently initiated using small DNA tiles with a size of 16 nm ϫ17 nm as folding staples. 26 Apart from these examples, there is no report for the preparation of larger origami structures (particularly 2D structures), and hence the development of new methods with added advantages is urgently required.In this work, we demonstrate a new route for the 2D extension of DNA origami using multiple JPs by programmed selfassembly, the spontaneous association of components into organized 2D structures using noncovalent interactions. By altering the shape of our previous origami structure, 25 we have designed nine different JPs, each of which is a 24-helix tile ( Figure 1A), containing (i) sequence-programmed connection sites, a tenon, and corresponding mortise, to allow assembly along the 10.1021/nn1031627© XXXX American Chemical Society ABSTRACT We demonstrate a novel strategy of self-assembly to scale up origami structures in twodimensional (2D) space using multiple origami structures, named "2D DNA jigsaw pieces", with a specially designed shape. For execution of 2D self-assembly along the helical axis (horizontal direction), sequence-programmed tenon and mortise were introduced to promote selective connections vi...